Peptides and related compositions and methods

ABSTRACT

Engineered peptides that bind with high affinity (low equilibrium dissociation constant (Kd)) to the cell surface receptors of fibronectin (α5β1) or vitronectin (αvβ3 and αvβ5 integrins) are disclosed as useful as imaging tissue. These peptides are based on a molecular scaffold into which a subsequence containing the RGD integrin-binding motif has been inserted. The subsequence (RGD mimic) comprises about 9-13 amino acids, and the RGD contained within the subsequence can be flanked by a variety of amino acids, the sequence of which was determined by sequential rounds of selection (in vitro evolution). The molecular scaffold is preferably based on a knottin, e.g., EETI (Trypsin inhibitor 2 (Trypsin inhibitor II) (EETI-II) [Ecballium elaterium (Jumping cucumber)], AgRP (Agouti-related protein), and Agatoxin IVB, which peptides have a rigidly defined three-dimensional conformation. It is demonstrated that EETI tolerates mutations in other loops and that the present peptides may be used as imaging agents.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under contract5K01CA104706 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the field of engineered peptides, andto the field of peptides which bind to integrins, and, particularly tointegrin binding as it relates to cell growth and development.

Related Art

Integrins are a family of extracellular matrix adhesion proteins thatnoncovalently associate into α and β heterodimers with distinct cellularand adhesive specificities (Hynes, 1992; Luscinskas and Lawler, 1994).Cell adhesion, mediated though integrin-protein interactions, isresponsible for cell motility, survival, and differentiation. Each α andβ subunit of the integrin receptor contributes to ligand binding andspecificity.

Protein binding to many different cell surface integrins can be mediatedthrough the short peptide motif Arg-Gly-Asp (RGD) (Pierschbacher andRuoslahti, 1984). These peptides have dual functions: They promote celladhesion when immobilized onto a surface, and they inhibit cell adhesionwhen presented to cells in solution. Adhesion proteins that contain theRGD sequence include: fibronectin, vitronectin, osteopontin, fibrinogen,von Willebrand factor, thrombospondin, laminin, entactin, tenascin, andbone sialoprotein (Ruoslahti, 1996). The RGD sequence displaysspecificity to about half of the 20 known integrins including the α₅β₁,α₈β₁, α_(v)β₁, α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈, and α_(iib)β3integrins, and, to a lesser extent, the α₂β₁, α₃β₁, α₄β₁, and α₇β₁integrins (Ruoslahti, 1996). In particular, the α_(v)β₃integrin iscapable of binding to a large variety of RGD containing proteinsincluding fibronectin, fibrinogen, vitronectin, osteopontin, vonWillebrand factor, and thrombospondin (Ruoslahti, 1996; Haubner et al.,1997), while the α₅β₁ integrin is more specific and has only been shownto bind to fibronectin (D'Souza et al., 1991).

The linear peptide sequence RGD has a much lower affinity for integrinsthan the proteins from which it is derived (Hautanen et al., 1989). Thisdue to conformational specificity afforded by folded protein domains notpresent in linear peptides. Increased functional integrin activity hasresulted from preparation of cyclic RGD motifs, alteration of theresidues flanking the RGD sequence, and synthesis of small moleculemimetics (reviewed in (Ruoslahti, 1996; Haubner et al., 1997)).

The X-ray crystal structure of the 10th type III domain of fibronectin(Dickinson et al., 1994), and the NMR solution structures of the murine9th and 10th type III fibronection domains (Copie et al., 1998)containing the RGD sequence have been solved. In these structures, theGRGDSP (SEQ ID NO: 105) amino acid sequence makes a type II β-hairpinturn that protrudes from the rest of the fibronectin structure forinteraction with integrin receptors.

Short RGD peptides also have been shown to assume a type II β-turn inaqueous solution, as determined by NMR (Johnson et al., 1993).Conformation and stereochemistry about the RGD motif in the form ofcyclic penta- and hexa-peptides, and disulfide-constrained peptides havebeen studied extensively (reviewed in (Haubner et al., 1997)). Previousapproaches have shown that combinations of natural and unnatural aminoacids, peptidomimetics, or disulfide bonds flanking the RGD motif havebeen necessary to create high affinity, biologically active β-turnstructures. The recent structure of an RGD β-loop mimic bound to 43(Xiong et al., 2002) has shed some interesting light on the nature ofthe ligand-receptor interaction and has validated the body of workencompassing the ligand-based design strategy.

Previously, phage display technology has been used to isolate cyclicpeptides specific to different integrin receptors. When a random linearhexapeptide library displayed on phage was panned with immobilizedintegrin, the amino acid sequence CRGDCL (SEQ ID NO: 1) was isolated(Koivunen et al., 1993). It was determined that this peptide was 10-foldmore potent than linear RGD hexapeptides in inhibiting the binding ofattachment of α₅β₁ expressing cells to fibronectin (Koivunen et al.,1993). This cyclic peptide also inhibited cell adhesion mediated byα_(v)β₁, α_(v)β3, and α_(v)β₅ integrins. In another study, phage displaywas used to isolate selective ligands to the α₅β₁ α_(v)β₃, α_(v)β₅, andα_(IIb) β₃ integrins from phage libraries expressing cyclic peptides(Koivunen et al., 1995). It was determined that each of the fourintegrins studied primarily selected RGD-containing sequences, butpreferred different ring sizes and flanking residues around the RGDmotif. A cyclic peptide, ACRGDGWCG (SEQ ID NO: 2), was isolated thatbound with high affinity to the α₅β₁ integrin. In addition, the cyclicpeptide ACDCRGDCFCG (SEQ ID NO: 3), which contains two disulfide bonds,was shown to be 20-fold more effective in inhibiting cell adhesionmediated by the α_(v)β₃ and α_(v)β₅ integrins than comparable peptideswith one disulfide bond, and 200-fold more potent than linear RGDpeptides.

Phage display has also been used to isolate novel integrin bindingmotifs from peptide libraries. The cyclic peptide CRRETAWAC (SEQ ID NO:4) was identified from a random heptapeptide phage library with flankingcystine residues (Koivunen et al., 1994). This peptide was specific forbinding to the α₅β₁ integrin, and not the α_(v)β₃ and α_(v)β₅ integrins,and was determined to have an overlapping binding site with the RGDsequence. The peptide NGRAHA (SEQ ID NO: 5) was identified by phagedisplay libraries as well (Koivunen et al., 1993), but it was laterdetermined that the receptor for this peptide was aminopeptidase N, andnot integrins as originally thought (Pasqualini et al., 2000). Asynergistic binding site on the 10th domain of fibronectin (encompassingthe sequence RNS) also enhances RGD binding to the α₅β₁ integrin(Koivunen et al., 1994; Obara and Yoshizato, 1995). In addition, thesequence PHSRN (SEQ ID NO: 6) (from the 9th domain of fibronectin),increases α₅β₁ integrin binding to the RGD peptide in fibronectin (Aotaet al., 1994). The sequence ACGSAGTCSPHLRRP (SEQ ID NO: 7) wasidentified from a 15-mer phage library panned with α_(v)β₃ integrin. TheSAGT (SEQ ID NO: 139) tetrapeptide is found in the sequence ofvitronectin, suggesting that this may be an accessory site for integrinrecognition and binding (Healy et al., 1995). It has been hypothesizedthat other synergy sites may exist (reviewed in Ruoslahti, 1996),suggesting that random peptide library screening for integrin ligandsother than RGD would be useful.

The presentation of multiple RGD motifs within one molecule has beenshown to increase integrin binding affinity and activity. Numerousstudies have demonstrated that multivalent clustering of RGD ligandswithin a polymer coated surface or bead results in enhanced celladhesion, due to increased local concentration of ligand, or increasedligand/receptor avidity. (Miyamoto et al., 1995; Maheshwari et al.,2000; Pierschbacher et al., 1994; Shakesheff et al., 1998). Soluble RGDrepeats incorporated into polypeptides (Saiki, 1997), or linked througha poly(carboxyethylmethacrylamide) backbone (Komazawa et al., 1993) havedemonstrated an increased potential for inhibition of cancer metastasiscompared to free peptide. More recently, soluble multivalent polymers ofGRGD (SEQ ID NO: 8), and copolymers of GRGD (SEQ ID NO: 8) and the α₅β₁synergy peptide SRN have been prepared synthetically throughring-opening metathesis (Maynard et al., 2001). Homopolymers containingGRGD (SEQ ID NO: 8) peptides were more potent inhibitors of fibronectincell adhesion (IC₅₀=0.18 mM) than peptide alone (IC₅₀=1.08 mM).Heteropolymers containing both GRGD (SEQ ID NO: 8) and SRN peptidesexhibited an enhanced ability to block fibronectin adhesion with an IC₅₀of 0.03 mM (Maynard et al., 2001). Although multivalent homo- andhetero-oligomers of integrin peptides demonstrated increased inhibitionof cell adhesion, improvements in affinity and efficacy are contemplatedthrough the use of multivalent frameworks.

The growth of new blood vessels, termed angiogenesis, plays an importantrole in development, wound healing, and inflammation (Folkman and Shing,1992). Angiogenesis has been implicated in proliferative disease statessuch as rheumatoid arthritis, cancer, and diabetic retinopathy, andtherefore is a relevant and attractive target for therapeuticintervention. In cancer, the growth and survival of solid tumors isdependent on their ability to trigger new blood vessel formation tosupply nutrients to the tumor cells (Folkman, 1992). With this new tumorvascularization comes the ability to release tumor cells into thecirculation leading to metastases. One specific approach toanti-angiogenic therapy is to inhibit cell adhesion events inendothelial cells. The α_(v)β₃ (Brooks et al., 1994) and α_(v)β₅integrins (Friedlander et al., 1995), and more recently the α₅β₁integrin (Kim et al., 2000), have been shown to be required forangiogenesis in vascular cells. Brooks and colleagues demonstrated thatthe α_(v)β₃integrin was abundantly expressed on blood vessels, but noton dermis or epithelial cells, and expression was upregulated onvascular tissue during angiogenesis (Brooks et al., 1994). In addition,the α_(v)β₁ integrin has been shown to be expressed on the tumorvasculature of breast, ovarian, prostate, and colon carcinomas, but noton normal adult tissues or blood vessels (Kim et al., 2000). The α_(v)β₃(and α_(v)β₅) integrins are highly expressed on many tumor cells such asosteosarcomas, neuroblastomas, carcinomas of the lung, breast, prostate,and bladder, as well as glioblastomas, and invasive melanomas (reviewedin (Haubner et al., 1997). It has also been demonstrated that theexpression levels of α_(v)β₃ and α_(v)β₅ by the vascular endothelium ofneuroblastoma was associated with the aggressiveness of the tumor(Erdreich-Epstein et al., 2000).

A monoclonal anti-α_(v)β₃ antibody (LM609) was shown to inhibitangiogenesis by fibroblast growth factor (FGF), tumor necrosis factor-a,and human melanoma fragments (Brooks et al., 1994). The humanizedversion of LM609, termed Vitaxin, has been shown to suppress tumorgrowth in animal models (Brooks et al., 1995), and target angiogenicblood vessels (Sipkins et al., 1998). Vitaxin has undergone Phase Iclinical trials in humans and appears to be safe and potentially activein disease stabilization (Gutheil et al., 2000). In another study,function-blocking anti-α₅β₁ monoclonal antibodies were shown to inhibitcell adhesion to fibronectin, and inhibit FGF-induced angiogenesis invivo (Kim et al., 2000). In addition, RGD peptides selective to α_(v)(Pasqualini et al., 1997) and α₅β₁ integrins (Kim et al., 2000) arerelevant targets for imaging and therapeutic purposes. Bacteriophagedisplaying an RGD peptide (CDCRGDCFC) (SEQ ID NO: 9) with high affinityto α_(v) integrins was shown to localize to tumor blood vessels wheninjected into tumor-bearing mice (Ruoslahti, 2000). In other approaches,RGD containing peptides and peptidomimetics have demonstrated promise incancer therapy by binding to overexpressed cell surface integrins andinterfering with angiogenesis and tumor blood supply. Inhibition ofα_(v)β₃ and α_(v)β₅ integrins by cyclic RGD peptides resulted insignificant reduction of functional blood vessel density, and was shownto impair tumor growth and metastasis in vivo (Brooks et al., 1994;Buerkle et al., 2002). In addition, the cyclic peptide c(RGDfV) (SEQ IDNO: 10) was shown to cause α_(v)β₃-mediated apoptosis in human malignantglioma cells (Chatterjee et al., 2000) and prostate cancer cells(Chatterjee et al., 2001). The cyclic peptide antagonist CRRETAWAC (SEQID NO: 11), and the nonpeptide antagonist SJ749, were shown toselectively inhibit a₅β₁-mediated cell adhesion to fibronectin, as wellas block FGF-induced angiogenesis in vivo (Kim et al., 2000). Ofparticular interest, the integrin inhibitors seem to have no effect onnormal vessels, and appear to function by specifically inducingapoptosis in newly budding endothelial cells during angiogenesis (Brookset al., 1994), and interfering with the function of metalloproteinaseenzymes required for cellular invasion (Brooks et al., 1996).

Radiolabeled integrin antagonists as described below are useful in tumortargeting and imaging applications. Noninvasive methods to visualize andquantify integrin expression in vivo are crucial for clinicalapplications of integrin antagonists (Brower, 1999). The firstgeneration of radioiodinated cyclic RGD peptides exhibited high affinityand specificity in vitro and in vivo for α_(v)β₃ integrins however,exhibited rapid excretion and accumulation in the liver and intestines,limiting their application (Haubner et al., 1999). Modifications ofthese peptides with a sugar moiety reduced their uptake in the liver,and increased their accumulation in α_(v)β₃-expressing tumors in vivo(Haubner et al., 2001). Noninvasive imaging with an ¹⁸F-labeled versionof this glycoRGD peptide by positron emission tomography demonstratedreceptor-specific binding and high tumor to background ratios in vivo,suggesting suitability for α_(v)β₃ quantification and therapy (Haubneret al., 2001). In addition, RGD peptides coupled to chelating agentscould be radiolabeled with ¹¹¹In ¹²⁵I, ⁹⁰Y, and ¹⁷⁷Lu, enlarging theirpotential for both tumor imaging and radionuclide therapy (van Hagen etal., 2000). Integrin-specific antibodies can also be useful for imagingapplications. Paramagnetic liposomes coated with the anti α_(v)β₃integrin antibody LM609 were used for detailed imaging of rabbitcarcinomas for a noninvasive means to asses growth and malignancy oftumors (Sipkins et al., 1998). The small integrin binding proteinsdescribed below would therefore be very amenable to coupling to avariety of radionuclides and chemotherapeutic agents.

Patents and Publications

Ruoslahti et al., have obtained a series of patents relating to RGDpeptides. For example, U.S. Pat. No. 5,695,997, entitled “Tetrapeptide,”relates to a method of altering cell attachment activity of cells,comprising: contacting the cells with a substantially pure solublepeptide including RGDX where X is any amino acid and the peptide hascell attachment activity. The patent further includes an embodimentwhere X is any amino acid and the peptide has cell attachment activityand the peptide has less than about 31 amino acids.

Similarly, U.S. Pat. No. 4,792,525 relates to a substantially purepeptide including as the cell-attachment-promoting constituent the aminoacid sequence Arg-Gly-Asp-R wherein R is Ser, Cys, Thr or other aminoacid, said peptide having cell-attachment promoting activity, and saidpeptide not being a naturally occurring peptide.

U.S. Pat. No. 5,169,930, to Ruoslahti, et al., relates to asubstantially pure integrin receptor characterized in that it consistsof an α_(v)β₁ subunit.

U.S. Pat. No. 5,536,814, to Ruoslahti, et al., entitled“Integrin-binding peptides,” issued Jul. 16, 1996, discloses a purifiedsynthetic peptide consisting of certain specified amino acid sequences.

U.S. Pat. No. 5,519,005, to Ofer et al., relates to certain non-peptidiccompounds comprising a guanidino and a carboxyl terminal groups with aspacer sequence of 11 atoms between them, which are effective inhibitorsof cellular or molecular interactions which depend on RXD or DGRrecognition, wherein X is G (gly), E (glu), Y (tyr), A (ala) or F (phe).These RXD and DGR analogues are referred to as “RXD surrogates.”

US 2005/0164300 to Artis, et al., published Jul. 28, 2005, entitled“Molecular scaffolds for kinase ligand development,” discloses molecularscaffolds that can be used to identify and develop ligands active on oneor more kinases, for example, the PIM kinases, (e.g., PIM-1, PIM-2, andPIM-3).

U.S. Pat. No. 6,451,976, to Lu et al., discloses a process in whichdendroaspin, a polypeptide neurotoxin analogue, is modified byrecombinant DNA techniques, particularly “loop grafting,” to provide amodified polypeptide.

U.S. Pat. No. 6,962,974, to Kalluri et al., issued Nov. 8, 2005,discloses recombinantly-produced Tumstatin, comprising the NCl domain ofthe α3 chain of Type IV collagen, having anti-angiogenic activity,anti-angiogenic fragments of the isolated Tumstatin, multimers of theisolated Tumstatin and anti-angiogenic fragments, and polynucleotidesencoding those anti-angiogenic proteins.

U.S. Pat. No. 5,766,591, to Brooks et al., relates to a method ofinducing solid tumor regression comprising administering anRGD-containing integrin αvβ3 antagonist.

U.S. Pat. No. 5,880,092 to Pierschbacher et al., relates to asubstantially pure compound comprising an Arg-Gly-Asp sequencestereochemically stabilized through a bridge and having a molecularweight less than about 5.4 kilodaltons.

U.S. Pat. No. 5,981,468 to Pierschbacher et al., relates to a compoundhaving a stabilized stereochemical conformation of a cyclic RGD peptide.

Koivunen et al., “Phage Libraries Displaying Cyclic Peptides withDifferent Ring Sizes: Ligand Specificities of the RGD-DirectedIntegrins,” Bio/Technology 13:265-270 (1995) discloses selective ligandsto the cell surface receptors of fibronectin (α₅β₁ integrin),vitronectin ((α_(v)β₃ integrin and α_(v)β₅ integrin and fibrinogen((α_(m)β₃integrin from phage libraries expressing cyclic peptides. Amixture of libraries was used that express a series of peptides flankedby a cystine residue on each side (CX5C, CX6C, CX7C) or only on one side(CX9) of the insert.

Reiss et al., “Inhibition of platelet aggregation by grafting RGD andKGD sequences on the structural scaffold of small disulfide-richproteins,” Platelets 17(3):153-7 (May 2006) discloses RGD and KGDcontaining peptide sequences with seven and 11 amino acids,respectively, which were grafted into two cystine knot microproteins,the trypsin inhibitor EETI-II and the melanocortin receptor bindingdomain of the human agouti-related protein AGRP, as well as into thesmall disintegrin obtustatin.

Wu et al., “Stepwise in vitro affinity maturation of Vitaxin, anα_(v)β₃-specific humanized mAb,” Proc. Nat. Acad. Sci. Vol. 95, Issue11, 6037-6042, May 26, 1998, discloses a focused mutagenesis implementedby codon-based mutagenesis applied to Vitaxin, a humanized version ofthe antiangiogenic antibody LM609 directed against a conformationalepitope of the α_(v)β₃ integrin complex. Wu et al., “Stepwise in vitroaffinity maturation of Vitaxin, an v3-specific humanized mAb,” Proc.Nat. Acad. Sci., Vol. 95, Issue 11, 6037-6042, May 26, 1998, discloses afocused mutagenesis implemented by codon-based mutagenesis applied toVitaxin, a humanized version of the antiangiogenic antibody LM609directed against a conformational epitope of the α_(v)β₃ integrincomplex.

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

In certain aspects, the present invention comprises an artificialintegrin binding peptide, based on a combination of a knottin peptideand an engineered loop, where the engineered loop provides a bindingsequence specific to bind to at least one of α_(v)β₅ integrin, α_(v)β₃integrin and α₅β₁ integrin, said binding sequence being comprised in aknottin protein scaffold. The knottin protein provides a “scaffold” dueto its relatively rigid three-dimensional structure. The bindingsequence will be an engineered integrin binding loop between 9 and 13amino acids long, said loop comprising the sequence RGD. Said scaffold,except for the engineered integrin binding loop, is identical or atleast substantially identical to one of: EETI-II, AgRP, mini-AGRP,agatoxin or miniagatoxin. It is shown here that certain scaffolds aretolerant to mutations in their loop regions.

In certain aspects, the present invention comprises an integrin bindingpeptide, comprising a binding sequence, which specifically binds to oneor both of α_(v)β5 and α_(v)β3 integrins. Certain sequences also bindonly to a₅β₁ integrin. It has been shown that some of the presentpeptides will bind to only α_(v)β₅ and α_(v)β3 integrins and not α₅β₁.The present engineered peptides further comprise a molecular scaffoldwhich is a knottin protein. As described, the knottin proteins arecharacterized by intramolecular bonds which stabilize them and form arigid scaffold. A portion of the scaffold, e.g., a loop beginning atresidue 3 of EETI-II, is replaced by a sequence that has beendiscovered, though in vitro molecular evolution, to have superiorbinding properties. The peptide thus has a scaffold comprisingreplacement of a portion of the knottin with an integrin binding loopbetween 9 and 13 amino acids long, said peptide substantially identicalto one of: EETI sequences as set forth in Table 2, AgRP sequences as setforth in Table 3 or mini-RGD-AgRP sequences as set forth in Table 4.

The present invention may further be characterized in that it comprisesan integrin binding peptide comprising a molecular scaffold, wherein themolecular scaffold is covalently linked to either end of an RGD mimicsequence, which is a loop consisting of about 8-12 amino acids, whichcomprise the sequence RGD, and preferably are selected from the groupconsisting of XXXRGDXXXXX (sequence (a)), 11 amino acids and XXRGDXXXX(sequence (b)), 9 amino acids, where X is any amino acid and said mimicsequence is linked at either end in the vicinity of, preferablyimmediately adjacent to, cross-linked residues, e.g., cysteines. Themolecular scaffold is preferably taken from a knottin peptide, and themimic sequence is inserted in the scaffold between the two cysteineresidues. The identity of the residues “X” can be varied in that,together, the X residues flanking the binding motif (RGD, RYD, etc.),provided a certain structure that will selectively recognize the ligand,in this case an endothelial integrin. Directed evolution techniques wereused and peptides with surprising selectivity and binding affinity wereobtained. It has been found that the number of residues on either sideof the RGD sequence is critical, particularly in relation to the threedimensional structure of the flanking Cys residues. That is, thelocation of RGD as after 3 residues and before 5 residues (sequence (a))is important with regard to the EETI scaffold, while the location insequence (b) is similarly important in the AgRP or agatoxin scaffold.The present EETI peptides will have 2-5 disulfide linkages betweencysteine residues, where the linkages are not directly between Cysresidues immediately flanking the RGD loop, as shown in FIG. 3. In otherknottins, there may be a disulfide linkage immediately flanking theloop, but in each case, there are at least two disulfide linkages,forming a molecular scaffold.

The present integrin binding peptides will have a specific affinity foran integrin selected from the group consisting of α₅β₁, α_(v)β₃ andα_(v)β₅, particularly α_(v)β₃. The present peptides preferably have a Kdless than 100 nM, or, more preferably less than 70 nM. Also theypreferably do not bind to integrin αIIbβ3, which is found on platelets.The term Kd means a dissociation constant, as is known in the art; lowerKd indicates tighter binding between the peptide and the integrin.

The molecular scaffold is preferably selected from the group consistingof EETI, AgRP, and agatoxin.

The sequences may be taken from a peptide having a sequencesubstantially identical to a peptide listed in Table 1 (EETI scaffoldcontaining native fibronectin loop), Table 2 (EETI mutant, RGD in loop4-6), Table 3 (AgRP peptides, RGD in loop) or Table 4 (mini-RGD-AgRPpeptides, RGD in loop). Substantial identity may be regarded as least70% identical, or at least 90-95% identical. Substantial identity may bedifferent in the RGD loop and in the knottin scaffold.

The peptides of the present invention can be made by recombinant DNAproduction techniques, including a vector encoding a peptide sequenceaccording to the present invention. The DNA sequences are chosenaccording to the genetic code, with codon preferences given according tothe host cell, e.g., mammalian, insect, yeast, etc. The present peptidesmay also be made by peptide synthetic methods.

Thus there is provided a method of inhibiting binding of an integrin tovitronectin and, in some cases, fibronectin, comprising contacting saidintegrin with an integrin binding peptide comprising a molecularscaffold, wherein the molecular scaffold is covalently linked to eitherend of an RGD mimic sequence selected from the group consisting ofXXXRGDXXXXX and XXRGDXXXX, where X is any amino acid. The presentinvention has been demonstrated with comparison to the 10^(th) domain offibronectin (“10FN” in the figures, e.g., FIGS. 4(f) and 4(i)).

Also provided is a method of treating a proliferative disease comprisingthe step of administering to a subject in need thereof a compositioncomprising an integrin binding peptide comprising a molecular scaffold,wherein the molecular scaffold is covalently linked to either end of anRGD mimic sequence selected from the group consisting of XXXRGDXXXXX andXXRGDXXXX, where X is any amino acid. A wide variety of proliferativedisorders will respond to the integrin inhibiting effects of the presentpeptides, which have been demonstrated with integrin α_(v)β₃, α_(v)β₅,and in some cases α₅β₁. For example, adhesive interaction of vascularcells through this integrin is known to be necessary for angiogenesis,and an antibody to this integrin has been shown to block angiogenesis.The present peptides may also be used in vitro or in vivo, e.g., in boneor tissue grafts, to promote cell adhesion by binding to cellsexpressing a selected integrin. The present peptides may also be used asimaging agents, in recognition of their affinity for integrins, whichare more highly expressed in certain types of cells. For example, tumorcells express higher levels of these integrins.

Also provided is a method for imaging tumors, in which engineeredintegrin binding peptides specific for certain integrins areadministered to a living organism, and the binding of the peptides tosites where endothelial integrins are highly expressed serves to imagetumors. The peptides disclosed here may be conjugated to a dye orradiolabel for such imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an example of flow cytometry data depicted as a dot plotof individual cells. Yeast cells are double-labeled with a labeledantibody against the c-myc epitope tag (x-axis), and ligand labeled withanother dye (y-axis). Since protein expression levels on the yeast cellsurface are variable, a ‘diagonal’ cell population results, in whichcells that express more protein bind more ligand; flow cytometry data isdepicted as a dot plot of individual cells.

FIG. 1B shows a schematic of the present yeast display system; yeastfusion proteins are expressed on the cell surface (Boder and Wittrup,1997). The yeast display construct shown in FIG. 1A has the generalorientation: Aga2-HA-engineered knottin-c-myc epitope, with the c-mycepitope at the carboxy terminus of the peptide. The displayed knottin islabeled with a chicken anti-cmyc antibody, which is then detected withan Alexa 555-labeled anti-chicken secondary antibody. The displayedknottin is allowed to bind to a test integrin. Bound integrin isdetected with an anti-integrin antibody, labeled with FITC.

FIG. 2 (left panel) is a schematic representation of an integrinantagonist having high specificity for one integrin (α_(v)β₃ here)engineered using yeast display and flow cytometry enrichment as referredto in FIG. 1. Integrin antagonists with ultra high specificity willallow for detection and inhibition of only certain integrins. FIG. 2(right panel) shows a high avidity integrin-binding protein in which theintegrin binding proteins described below are presented in a tetravalentmanner through linkage to a GCN 4-zipper, which spontaneouslyself-assembles to form a tetramer. Tetravalent presentation of theintegrin antagonists will enhance integrin binding by increasing thelocal concentration of antagonist, upon binding of the first antagonist.

FIGS. 3A, 3B and 3C show the positions of the Cys-Cys disulfide linkagesin the sequences of knottin proteins EETI-II (SEQ ID NO: 13), AgRP (SEQID NO: 107) and omega agatoxin 4B (SEQ ID NO: 16). Cysteine residues canbe seen to be immediately flanking the RGD mimic loops, which, in thepresent engineered peptides, are between the brackets. For example, inAgRP, it can be seen that the cysteines flanking the RGD mimic sequencewill be linked to each other, whereas in EETI they are not. The size ofthe grafted sequence will depend on the molecular framework structure,such that shorter loops will be preferred in cases where they are in theframework adjacent linked cysteines. Other loops between Cys residuesmay be engineered according to the present methods. Disulfide linkagesfor other knottin proteins are set forth in the knottin database. FIG.3A-C is adapted from Biochemistry, 40, 15520-15527 (2001) and J. Biol.Chem., 2003, 278:6314-6322.

FIG. 4A-FIG. 41 is a series of nine panels, (a) through (i) from topleft to bottom right, showing flow cytometry data obtained foryeast-displayed RGD-EETI #3 (also called FN-RGD). Panels (a) through (c)are controls; FL1-H represents the signal generated from theFITC-labeled integrin antibody, and FL2-H represents the signalgenerated from the chicken anti-cmyc antibody+Alexa-555 labeledanti-chicken secondary antibody. Panels (d) through (f) are histogramsof the data presented below in panels (g) (h) and (i). Panels (g), (h)and (i) (Bottom row) are dot plots of RDG-EETI #3 induced at 30° C.,with 100 nM integrin α_(v)β₃(e), at 20° C., with 100 nM integrinα_(v)β₃(f) and 10FN (10^(th) domain of fibronectin) induced at 30° C.,with 100 nM integrin α_(v)β₃. The plots show that the RGD-EETI #3(FN-RGD) peptide binds better than the 10^(th) domain of fibronectin, anatural α_(v)β₃ integrin binder, and, further, that the peptide foldscorrectly at both 30° C. and 20° C. expression.

FIG. 5A through FIG. 5G is a series of seven panels, (a) through (g)from top left to bottom right, showing flow cytometry data obtained foryeast-displayed RGD-AgRP #3 (panel d), Agatoxin #2 (panel e), mini AgRP(panel f) and mini-RGD-Agatoxin (panel g). Panels (a) through (c) arecontrols; parameters are FL1-H and FL2-H are as in FIG. 4; Second rowpanels (d) and (e) are, respectively, dot plots of RGD-AgRP #3 with 100nM integrin α_(v)β₃ and RGD-agatoxin #2 with 100 nM integrin α_(v)β₃.Third row panels (f) and (g) are, respectively, dot plots ofmini-RGD-AgRP with 100 nM integrin α_(v)β₃ and mini-RGD-agatoxin with100 nM integrin α_(v)β₃. The plots show that the “mini” versions ofRGD-AgRP #3 and RGD-agatoxin#2 bind to integrin α_(v)β₃ just as well asthe full-length versions.

FIG. 6A through FIG. 6M is a series of 13 panels (a) through (m) showingdot plots of EETI-based RGD mutants obtained by directed evolution,labeled with 100 nM of α_(v)β₃ integrin. The first row consists ofcontrols. FL1H and FL2H are labeled as before. The samples are labeledfrom left to right for each row. Samples 1.5B (d), 1.4B (e), 1.5F (f),2.4F (g), 2.5A (h), 2.5C (i), 2.5D (j), 2.5F (k), 2.5H (1) and 2.5J (m)represent EETI-based variants as set forth in Table 2.

FIG. 7A through FIG. 7M is a series of 13 panels showing dot plots andhistograms (panels (c), (f), (i), (k), and (m)) showing a control (a),and the samples as labeled in the center column of the figure, i.e.,1.5B, 2.4F, 2.5A, 2.5D and 2.5J. These peptides are labeled with 50 nMintegrin α_(v)β₃ and further defined in the table below. The flowcytometry parameters are as given above. FIG. 7 shows that the bestmutants appear to be 1.5B, 2.5A, and 2.5D. This data suggests Kd valuesof about 50 nM. When displayed on the yeast cell surface, these mutantsbind to α_(v)β₃ integrin about 2-3× better than the starting mutantRGD-EETI #3 (FN-RGD), although this is a gross estimate since we did nothave enough soluble α_(v)β₃ integrin to perform full titration curves.

FIG. 8A is a series of 4 pictures showing in vivo imaging ofCy5.5-labeled polypeptides in mice. 1.5 nmol of Cy5.5-labeled EETI-RGDpeptide 2.5D or other indicated peptide was injected by tail vein intoU87MG glioblastoma xenograft mouse models and imaged at various timepoints post injection. Arrows indicate the position of tumors. FIG. 8Bis a graph showing quantified tumor/normal tissue ratio forCy5.5-labeled 2.5D (top line, triangles) compared to Cy5.5-labeledFN-RGD (middle line, squares) and Cy5.5-labeled c(RGDyK) (SEQ ID NO:140) (middle line, circles). The tumor/background ratio shows ˜60%greater contrast for the high affinity evolved 2.5D peptide over theweaker binding FN-RGD and c(RGDyK) (SEQ ID NO: 140) peptides.Cy5.5-labeled FN-RDG negative control (bottom line, open squares)indicates background levels. FIG. 8C is a series of images of differentorgans showing uptake of Cy5.5-labeled 2.5D and a comparison peptide,c(RGDyK) (SEQ ID NO: 140). It can be seen that the tumor took upsignificantly more 2.5D than c(RGDyK) (SEQ ID NO: 140), and that otherorgans were not significantly showing fluorescence, except for thekidney, where the peptide would accumulate prior to excretion.

FIG. 9 is a graph showing normalized competition plotted against peptideconcentration in an integrin-binding assay on U87MG glioblastoma cells.Relative polypeptide binding affinity was measured by competition of¹²⁵I-labeled echistatin with unlabeled echistatin (line 1), 2.5D (line2), FN-RGD (line 5), c(RGDyK) (SEQ ID NO: 140) (line 4), and scrambledFN-RDG (line 3).

FIG. 10A through FIG. 10D is a series of histograms showing bindingspecificities to integrins α_(v)β₃, α_(v)β₅, α₅β₁, and α_(iib)β₃ forengineered EETI-RGD peptides compared to controls. Error bars representexperiments performed in triplicate. Competition binding of 0.06 nM ¹²⁵Iechistatin with 5 nM (black bars) and 50 nM (grey bars) unlabeledpeptide to plate-coated integrins was measured. 1=echistatin; 2=c(RGDyK)(SEQ ID NO: 140); 3=FN-RGD; 4=1.5B; 4=2.5D; 5=2.5F; 6=FN-RDG. Theengineered peptides have very little binding to α_(iib)β₃ integrin.

FIG. 11 is a series of histograms showing residue distribution ofmutants isolated from EETI XXXRGDXXXXX library #2. The distribution ofresidues in different positions is shown for each position.

FIGS. 12A and 12B are a pair of bar graphs showing binding results ofmutagenesis of AgRP loops 1-3 using degenerate codons. Binding of 50 nMintegrin to yeast-displayed AgRP peptide clones from sort round 4 ofdegenerate codon libraries is shown. Top graph shows binding to α_(v)β₃integrin; bottom graph shows binding to α_(iib)β₃ integrin. Background,marked “bg” indicates cells stained with fluorescein-conjugatedanti-integrin antibodies only. Numbers are arbitrary fluorescence units.

FIG. 13 is a chart showing radioactivity accumulation quantification inselected organs of U87-MG tumor bearing mice at 30 min, 1 hour, 2 hour,4 hour and 24 hour after injection of ⁶⁴Cu-DOTA-7C AgRP engineeredpeptide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Overview

The present invention involves the selection of a knottin protein as apeptide framework (scaffold) and replacing a portion of the sequencethat appears on the surface with a specific binding sequence, e.g.,containing an integrin binding sequence (RGD). The resulting engineeredpeptides have high affinity and specificity for selected integrinspresent on surfaces of tumor cells, epithelial cells, and the like.

Directed evolution is a useful technology for creating novelbiomolecules that enhance or mimic protein function. Small polypeptideswith applications as therapeutics and research tools were developedusing directed evolution. These peptides are amenable to chemicalsynthesis and offer facile incorporation into biomaterials. Usingmolecular cloning, biologically active amino acid sequences derived fromcell adhesion proteins (fibronectin) were grafted into several stable,constrained knottin peptide frameworks (EETI, AgRP and Agatoxin IVB) andwere shown to bind to integrin receptors (α_(v)β₃) with modest affinity.Since polypeptide conformation is critical for high affinity receptorbinding and specificity, prototype molecules were subjected to affinitymaturation using molecular evolution. Combinatorial libraries of mutantsdisplayed on the yeast cell surface were screened by flow cytometricsorting to isolate polypeptides with enhanced integrin binding affinity.These proteins specifically modulate integrin-mediated cell adhesion andcan serve as molecular imaging agents. These results demonstrate thatnaturally occurring constrained peptide scaffolds 1) can be redirectedto function as adhesion molecule mimics and 2) can be engineered forenhanced integrin binding affinity through directed evolution.

The present methods have led to the development of specific bindingpeptides against the α_(v)β₃, α_(v)β₅, and, in some cases, the α5β₁integrin receptors, which have been implicated in cell adhesion andangiogenesis of vascular tissue in cancer. Integrin-specific binderscomprised of the cyclic peptide Arg-Gly-Asp (RGD, discussed inBackground) have shown much therapeutic promise, but can benefit fromimprovements in affinity and stability. A novel selection approach basedon yeast surface display was utilized for affinity maturation andstabilization of molecular scaffolds containing the RGD motif. Inaddition, frameworks for multivalent RGD ligand presentation throughchemical crosslinking and protein engineering are presented.

Knottin proteins containing RGD motifs were assayed for binding againstintegrins. It was found that the scaffolds offer an extremely stableplatform for conformationally constrained ligand presentation and are auseful framework for protein engineering studies. In addition,multivalent protein scaffolds can be engineered by replacing multiplebinding faces of knottin proteins with RGD motifs for enhanced integrinbinding.

Multivalent presentation of integrin-specific motifs through chemicalcrosslinking is also contemplated here. Receptor clustering has beenshown to be important for high avidity integrin binding and function. Aseries of crosslinking agents can be developed for multivalent integrinligand presentation using novel coupling methodology. Synergisticeffects have been shown to exist between RGD and other integrin-specificpeptide motifs. Therefore, cross linkers could also be designed thatincorporate heterofunctional groups to couple differentintegrin-specific molecules. These multivalent integrin binding proteinsand peptides can be tested for their ability to enhance integrin bindingand antagonism of cell adhesion.

Combinatorial mutant libraries of RGD-based knottin scaffolds expressedon yeast were screened for specific, high affinity binding againstsoluble α_(v)β₃ integrin using flow cytometry.

Definitions

The term “molecular scaffold” means a polymer having a predefinedthree-dimensional structure, into which can be incorporated a bindingloop, which will contain an RGD mimic as described herein. The term“molecular scaffold” has an art-recognized meaning (in other contexts),which is also intended here. For example, a review by Skerra,“Engineered protein scaffolds for molecular recognition,” J. Mol.Recognit. 2000; 13:167-187 describes the following scaffolds: singledomains of antibodies of the immunoglobulin superfamily, proteaseinhibitors, helix-bundle proteins, disulfide-knotted peptides andlipocalins. Guidance is given for the selection of an appropriatemolecular scaffold.

Incorporation of integrin binding motifs into a molecular (e.g.,protein) scaffold offers a framework for ligand presentation that ismore rigid and stable than linear or cyclic peptide loops. In addition,the conformational flexibility of small peptides in solution is high,and results in large entropic penalties upon binding. Incorporation ofan RGD motif into a protein scaffold provides conformational constraintsthat are required for high affinity integrin binding, (as evidenced bythe CDCRGDCFC (SEQ ID NO: 12) peptide described above (Koivunen et al.,1995)). Furthermore, the scaffold provides a platform to carry outprotein engineering studies such as affinity or stability maturation.

Characteristics of a desirable scaffold for protein design andengineering include 1) high stability in vitro and in vivo, 2) theability to replace amino acid regions of the scaffold with othersequences without disrupting the overall fold, 3) the ability to createmultifunctional or bispecific targeting by engineering separate regionsof the molecule, and 4) a small size to allow for chemical synthesis andincorporation of non-natural amino acids if desired. Scaffolds derivedfrom human proteins are favored for therapeutic applications to reducetoxicity or immunogenicity concerns, but are not always a strictrequirement. Other scaffolds that have been used for protein designinclude fibronectin (Koide et al., 1998), lipocalin (Beste et al.,1999), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (Hufton etal., 2000), and tendamistat (McConnell and Hoess, 1995; Li et al.,2003). While these scaffolds have proved to be useful frameworks forprotein engineering, molecular scaffolds such as knottins have adistinct advantage: their small size.

The term “proliferative diseases” refers to diseases in which sometissue in a patient proliferates at a greater than normal rate.Proliferative diseases may be cancerous or non-cancerous. Non-cancerousproliferative diseases include epidermic and dermoid cysts, lipomas,adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevilesions, teratomas, nephromas, myofibromatosis, osteoplastic tumors,other dysplastic masses and the like.

The types of proliferative diseases which may be treated or imaged withcompounds and compositions of the present invention include epidermicand dermoid cysts, lipomas, adenomas, capillary and cutaneoushemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas,myofibromatosis, osteoplastic tumors, other dysplastic masses and thelike.

The types of cancers which may be treated or imaged with compounds andcompositions of the present invention include: breast carcinoma, bladdercarcinoma, brain cancer, colorectal carcinoma, esophageal carcinoma,gastric carcinoma, germ cell carcinoma e.g., testicular cancer,gynecologic carcinoma, hepatocellular carcinoma, small cell lungcarcinoma, non-small cell lung carcinoma, lymphomas, Hodgkin's lymphoma,non-Hodgkin's lymphoma, malignant melanoma, multiple myeloma, neurologiccarcinoma, ovarian carcinoma, pancreatic carcinoma, prostate carcinoma,renal cell carcinoma, Ewings sarcoma, osteosarcoma, soft tissue sarcoma,pediatric malignancies and the like.

The term “effective amount” means an amount of a compound of the presentinvention that is capable of modulating binding of an integrin to acognate ligand.

The term “knottin protein” means a structural family of small proteins,typically 25-40 amino acids, that bind to a range of molecular targetslike proteins, sugars and lipids. Their three-dimensional structure isessentially defined by a peculiar arrangement of three to five disulfidebonds. A characteristic knotted topology with one disulfide bridgecrossing the macro-cycle limited by the two other intrachain disulfidebonds, which was found in several different microproteins with the samecysteine network, lent its name to this class of biomolecules. Althoughtheir secondary structure content is generally low, the knottins share asmall triple-stranded antiparallel β-sheet, which is stabilized by thedisulfide bond framework. Biochemically well-defined members of theknottin family, also called cysteine knot proteins, include the trypsininhibitor EETI-II from Ecballium elaterium seeds, the neuronal N-typeCa²⁺ channel blocker ω-conotoxin from the venom of the predatory conesnail Conus geographus, agouti-related protein (See Millhauser et al.,“Loops and Links: Structural Insights into the Remarkable Function ofthe Agouti-Related Protein,” Ann. N.Y. Acad. Sci., Jun. 1, 2003; 994(1):27-35), the omega agatoxin family, etc.

Knottin proteins are shown in FIG. 3 as having a characteristicdisulfide linking structure. This structure is also illustrated in Gellyet al., “The KNOTTIN website and database: a new information systemdedicated to the knottin scaffold,” Nucleic Acids Research, 2004, Vol.32, Database issue D156-D159. A triple-stranded β-sheet is present inmany knottins. The cysteines involved in the knot are shown as connectedby lines in FIG. 3 indicating which Cys residues are linked to eachother. The spacing between Cys residues is important in the presentinvention, as is the molecular topology and conformation of theRGD-containing integrin binding loop. These attributes are critical forhigh affinity integrin binding. The RGD mimic loop is inserted betweenknottin Cys residues, but the length of the loop must be adjusted foroptimal integrin binding depending on the three-dimensional spacingbetween those Cys residues. For example, if the flanking Cys residuesare linked to each other, the optimal loop may be shorter than if theflanking Cys residues are linked to Cys residues separated in primarysequence. Otherwise, particular amino acid substitutions can beintroduced that constrain a longer RGD-containing loop into an optimalconformation for high affinity integrin binding.

The term “amino acid” includes both naturally occurring and syntheticamino acids and includes both the D and L form of the acids as well asthe racemic form. More specifically, amino acids contain up to tencarbon atoms. They may contain an additional carboxyl group, andheteroatoms such as nitrogen and sulfur. Preferably the amino acids areα and β-amino acids. The term α-amino acid refers to amino acids inwhich the amino group is attached to the carbon directly attached to thecarboxyl group, which is the α-carbon. The term β-amino acid refers toamino acids in which the amino group is attached to a carbon one removedfrom the carboxyl group, which is the β-carbon. The amino acidsdescribed here are referred to in standard IUPAC single letternomenclature, with “X” meaning any amino acid.

The term “EETI” means Protein Data Bank Entry (PDB) 2ETI. Its entry inthe Knottin database is EETI-II. It has the sequence

(SEQ ID NO: 13) GC  PRILMR  [CKQDSDC]LAGCV[CGPNGFC]G.

The bold and underlined portion is replaced above and in the examplesbelow by the present RGD mimic sequence(s). Loops 2 and 3, including theend defining cysteines, are show in brackets. These loops can also bevaried without affecting binding efficiency, as is demonstrated below.

The term “AgRP” means PDB entry 1HYK. Its entry in the Knottin databaseis SwissProt AGRP_HUMAN, where the full-length sequence of 129 aminoacids may be found. It comprises the sequence beginning at amino acid87. An additional G is added to this construct. It also includes a C105Amutation described in Jackson, et al. 2002 Biochemistry, 41, 7565.

(SEQ ID NO: 14) GCVRLHESCLGQQVPCCDPCATCYC RFFNAF CYCR-KLGTAMNPCSRT

The dashed portion shows a fragment omitted in the “mini” version,below. The bold and underlined portion, from loop 4, is replaced by theRGD sequences described below.

The term “mini” in reference to AgRP means PDB entry 1MRO. It is alsoSwissProt AGRP_HUMAN. It has the sequence, similar to that given above,

(SEQ ID NO: 15) GCVRLHESCLGQQVPCCDPAATCYC RFFNAF CYCR

where the italicized “A” represents an amino acid substitution whicheliminates a possible dimer forming cystine. (Cystine herein refers tothe single amino acid; cysteine to the dimer.). The bold and underlinedportion, from loop 4, is replaced by the below described he RGDsequences.

The term “agatoxin” means omega agatoxin PDB 10 MB and the SwissProtentry in the knottin database TOG4B_AGEAP. It has the sequence

(SEQ ID NO: 16) EDN--CIAEDYGKCTWGGTKCCRGRPCRC SMIGTN CECT-PRLIMEGLS FA

The dashes indicate portions of the peptide omitted for the “mini”agatoxin. As shown in Table 3, an additional glycine is added to theN-terminus of the mini-construct. The bold and underlined portion isreplaced by the below described he RGD sequences.

The term “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70% sequence identityto a reference sequence, preferably 80%, more preferably 85%, mostpreferably at least 90% or at least 95% sequence identity to thereference sequence over a specified comparison window, which in thiscase is either the entire peptide, a molecular scaffold portion, or abinding loop portion (˜9-11 residues). Preferably, optimal alignment isconducted using the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol., 48:443 453. An indication that two peptidesequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Another indication for present purposes, that a sequence issubstantially identical to a specific sequence explicitly exemplified isthat the sequence in question will have an integrin binding affinity atleast as high as the reference sequence. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution. “Conservativesubstitutions” are well known, and exemplified, e.g., by the PAM 250scoring matrix. Peptides that are “substantially similar” sharesequences as noted above except that residue positions that are notidentical may differ by conservative amino acid changes. As used herein,“sequence identity” or “identity” in the context of two nucleic acid orpolypeptide sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window. When percentage of sequence identity isused in reference to proteins it is recognized that residue positionswhich are not identical often differ by conservative amino acidsubstitutions, where amino acid residues are substituted for other aminoacid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. When sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the NIH Multiplealignment workshop (http://helixweb.nih.gov/multi-align/).Three-dimensional tools may also be used for sequence comparison.

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “endothelial integrin” is used in its conventional sense andmeans integrins expressed on the outer apical pole of the surfaceepithelium, and are involved in angiogenesis. More specific details arefound at J. Clin. Invest., 110:913-914 (2002).

The term “optical label” is used in its conventional sense to mean,e.g., Cy-5.5 and other dyes useful as near infrared imaging agents. Avariety of optical labels can be used in the practice of the inventionand include, for example,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumarin 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodarnine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);Cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; La Jolta Blue;phthalo cyanine; and naphthalo cyanine. Other useful labels include theAlexa Fluor® dyes from Invitrogen, which are sulfonated dyes, based onaminocoumarin, rhodamine, etc.

The term “positron-emitting label” is used in its conventional sense andmeans a label for detection by a positron emission camera, as inpositron emission tomography, in which the label is attached, e.g., viaa chelator, to a peptide according to the present invention. The mostcommon labels used positron emitting nuclei in PET are ¹¹C, ¹³N, ¹⁵O and¹⁸F. Positron emitters zirconium-89 (⁸⁹Zr) and iodine-124 (¹²⁴I) arealso contemplated for their long half life. Other labels include inparticular ^(94m)Tc, ⁶⁸Ga and ¹⁸F, ⁶⁴Cu, ⁸⁶Y, and ⁷⁶Br.

The term “engineered integrin binding loop” means a primary sequence ofabout 9-13 amino acids which have been created ab initio throughexperimental methods such as directed molecular evolution to bind toendothelial integrins. That is, the sequence contains an RGD sequence orthe like, placed between amino acids which are particular to thescaffold and the binding specificity desired. The RGD (RYD, etc) bindingsequence is not simply taken from a natural binding sequence of a knownprotein.

EXPERIMENTAL Library Creation

In order to generate a randomized library of RGD mimic sequences,oligonucleotides were prepared which coded for various RGD mimicsequences as they were to be contained within a selected knottinscaffold. Since the knottin/RGD engineered sequence was relativelyshort, the DNA used to express the engineered protein in yeast could beprepared synthetically. The DNA sequences to be ligated into the yeastdisplay vector were obtained from MWG-BIOTECH Inc., High Point, N.C.Where an amino acid was to be varied, twenty different codons, eachcoding for a different amino acid, were synthesized for a givenposition. Randomized oligonucleotide synthesis has been used to create acoding cassette in which about 5 to about 15 amino acids are randomized(see, e.g., Burritt et al., (1996) Anal. Biochem. 238:1 13; Lowman(1997) Annu. Rev. Biophys. Biomol. Struct. 26:410 24; Wilson (1998) Can.J. Microbiol. 44:313 329).

The yeast display vector used for evolution of improved mutants iscalled “pCT”. The vector is further described in US 2004/0146976 toWittrup, et al., published Jul. 29, 2004, entitled “Yeast cell surfacedisplay of proteins and uses thereof.” As described there, the vectorprovides a genetic fusion of the N terminus of a polypeptide of interestto the C-terminus of the yeast Aga2p cell wall protein. The outer wallof each yeast cell can display approximately 10⁴-10⁵ proteinagglutinins. The vector contains the specific restriction sites andillustrates the transcriptional regulation by galactose, the N-terminalHA and C-terminal c-myc epitope tags and the Factor Xa protease cleavagesite.

The vector used in the present work contained NheI (GCTAGC) (SEQ ID NO:17) and BamHI (GGATCC) (SEQ ID NO: 18) restriction sites for specificinsertion of the RGD mimic coding sequence.

Labeling Yeast-Displayed Polypeptides

Below is a typical protocol to label a yeast library samples for sortingby flow cytometry (FACS):

-   -   1. Want 2×10⁶ cells, OD₆₀₀ of 1.0≈10⁷cells/mL    -   2. Add 1 mL PBS/BSA (phosphate buffered saline containing 1        mg/mL bovine serum albumin) to wash cells    -   3. Spin down cells 3 min at 8000 RPM    -   4. Remove supernatant using vacuum    -   5. Re-suspend in 40 μL PBS/BSA containing proper amount of        integrin (100 nM [2.5 μL of stock α_(v)β₃]; no anti-cmyc at this        point)    -   6. Incubate for 1.5 h at r.t (w/tumbling)    -   7. Add 1:250 dilution of (chick anti-cmyc) to labeling solution    -   8. DO NOT wash cells at this point.    -   9. Incubate 1 h at 4° C. (w/tumbling)    -   10. Keep on ice after this step.    -   11. Spin down cells 3 min at 8000 RPM, 4° C. and vacuum        supernatant    -   12. Repeat wash steps 2-4    -   13. Re-suspend in 40 μL PBS/BSA containing proper amount of        secondary labels (secondary labeling is simultaneous)        -   i. Anti-integrin Ab (FITC conj): 1:25 dilution+Anti-chick            (Alexa 555): 1:100 dilution        -   ii. Positive control: Anti-chick (Alexa 555): 1:100 dilution            (for FACS compensation)        -   iii. Positive control: Anti-chick (Alexa 488): 1:100            dilution (for FACS compensation)    -   14. Incubate on ice 30 min and keep in dark (lid on ice bucket)    -   15. Spin down cells at 3 min at 8000 RPM, 4° C. and vacuum        supernatant    -   16. Repeat steps 2-4: Add 1 mL PBS/BSA, pellet cells, vacuum        supernatant    -   17. Leave pelleted cells on ice until use.

Fluorescent Cell Sorting

Commercially available flow cytometers can measure fluorescenceemissions at the single-cell level at four or more wavelengths, at arate of approximately 50,000 cells per second (Ashcroft and Lopez,2000). Typical flow cytometry data are shown in FIG. 4-7, in which yeasthave been labeled with two different color fluorescent probes to measureprotein expression levels and bound soluble ligand (in this caseintegrin receptor). A ‘diagonal’ population of cells results due tovariation in protein expression levels on a per cell basis: cells thatexpress more protein will bind more ligand. The equilibrium bindingconstant (K_(d)) can be determined by titration of soluble ligand, andthe dissociation rate constant (k_(off)) can be measured throughcompetition binding of unlabeled ligand. With yeast, a monodispersity oftethered proteins exists over the cell surface, and soluble ligand areused for binding and testing, such that avidity effects are notobserved, unlike other display methods using immobilized ligands. Todate, the properties of most proteins expressed on the yeast cellsurface mimic what is seen in solution in terms of stability and bindingaffinity (Bader et al., 2000; Feldhaus et al., 2003; Holler et al.,2000; VanAntwerp and Wittrup, 2000). See, also, Weaver-Feldhaus et al.,“Directed evolution for the development of conformation-specificaffinity reagents using yeast display,” Protein Engineering Design andSelection Sep. 26, 2005 18(11):527-536.

Cell sorting was carried out on a FACSVantage (BD Biosciences)multiparameter laser flow cytometer and cell sorter. Before sorting,fluorescent staining was carried out as described above, so thatanalysis of integrin binding and c-myc expression levels were detected,as described above. Cells with the highest levels of integrin binding,normalized for c-myc expression levels, were gated and sorted into acollection tube containing culture media. Sorted clones were propagatedin culture and flow cytometric screening was repeated several times toobtain an enriched population of yeast-displayed peptides with highaffinity integrin binding.

After obtaining a pool of cells with high integrin binding affinity,single yeast clones were obtained by plating onto Petri dishes. PlasmidDNA was obtained from the entire yeast population, transformed to E.coli and then individual E. coli clones were selected for plasmidrecovery and sequencing to determined the aa composition of individualmutants. The DNA sequences of representative peptides are given below.

Sequence Design: EETI-II Scaffold and Mini-AgRP Scaffold

The sequences listed below were generated from three different yeastdisplayed combinatorial libraries—two libraries based on the EETI-IIscaffold and one library based on the Mini-AgRP scaffold. All librarieswere sorted by fluorescence activated cell sorting (FACS). Mutants fromeach round were isolated and sequenced.

The EETI-II-based library was:

(SEQ ID NO: 19) GC XXXRGDXXXXX CKQDSDCLAGCVCGPNGFCG,where X=any amino acid. This library produced mutants 1.x, listed below.A follow-up library was made in a similar manner in an attempt toimprove the mutants made from the original library just mentioned. Theseresultant mutants are labeled 2.x, listed below.

In the above sequence EETI-II loops 2 and 3 (from left to right) arealso underlined, but not bolded. Flanking cysteines are not underlined.As will be discussed below, these sequences have been shown to betolerant to diversity without affecting the binding capacity of thebinding loop.

The above-described library was also prepared with the insert XXRGDXXXXand these EETI-II peptides were mixed with the library shown here beforesorting. However, no sequences from this library were isolated. Thisindicates the importance of having the proper number of flankingresidues around the RGD sequence in this scaffold.

EETI-II Based Sequences:

TABLE 1 Sequences wherein the RGD motif (in italics)is found in the insert at positions 3-5. Peptide identifier SequenceSEQ ID NO: RGD-EETI#2 GCTG

SPASSKCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 20) RGD-EETI#2 GCVTG

SPASSCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 21)

RGD-EETI #3 had binding estimated to be in the range of Kd 100-200 nM.RGD-EETI #2 had approximately half the affinity of RGD-EETI #3. Thebolded sequences were chosen for initial loop design from nativefibronectin RGD loop sequences.

Variants based on RGD-EETI #3 above were prepared, with the RGD motif atamino acid positions 6-8, where the sequence RGD is italicized in 1.4A.In other words, the starting library wasGCXXXRGDXXXXXCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 22)

The integrin-binding loop was inserted after the second residue and thefirst Cys.

TABLE 2 EETI sequences wherein the RGD motif (in italics in 1.4A)is found in the insert at positions 4-6. Peptide identifier SequenceSEQ ID NO: 1.4A GC AEP

MPWTW CKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 23) 1.4B GC VGGRGDWSPKWCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 24) 1.4C GC AELRGDRSYPECKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 25) 1.4E GC RLPRGDVPRPHCKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 26) 1.4H GC YPLRGDNPYAACKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 27) 1.5B GC TIGRGDWAPSECKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 28) 1.5F GC HPPRGDNPPVTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 29) 2.3A GC PEPRGDNPPPSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 30) 2.3B GC LPPRGDNPPPSCKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 31) 2.3C GC HLGRGDWAPVGCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 32) 2.3D GC NVGRGDWAPSECKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 33) 2.3E GC FPGRGDWAPSSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 34) 2.3F GC PLPRGDNPPTECKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 35) 2.3G GC SEARGDNPRLSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 36) 2.3H GC LLGRGDWAPEACKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 37) 2.3I GC HVGRGDWAPLKCKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 38) 2.3J GC VRGRGDWAPPSCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 39) 2.4A GC LGGRGDWAPPACKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 40) 2.4C GC FVGRGDWAPLTCKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 41) 2.4D GC PVGRGDWSPASCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 42) 2.4E GC PRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 43) 2.4F GC YQGRGDWSPSSCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 44) 2.4G GC APGRGDWAPSECKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 45) 2.4J GC VQGRGDWSPPSCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 46) 2.5A GC HVGRGDWAPEECKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 47) 2.5C GC DGGRGDWAPPACKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 48) 2.5D GC PQGRGDWAPTSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 49) 2.5F GC PRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 50) 2.5H GC PQGRGDWAPEWCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 51) 2.5J GC PRGRGDWSPPACKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 52)

Thus there has been described an engineered integrin binding peptidecomprising a scaffold sequence and an RGD insert, both of which may bemodified as described.

The EETI-II scaffold as described above reveals a number of specificsequences, which form the EETI-II knottin scaffold as illustrated inFIG. 3, having three disulfide linkages. The native sequence, which isreplaced by the insert is shown in brackets in FIG. 3 and in boldunderline above; the insert is shown in bold underline in Tables 1 and2. The scaffold sequence may be varied, as the primary function of thescaffold is to maintain the orientation of the RGD insert. The scaffoldshould have the GPNG (SEQ ID NO: 109) sequence, which is known to beneeded for folding (Wentzel, et al., “Sequence Requirements of the GPNG(SEQ ID NO: 109) β-Turn of the Ecballum elaterium Trypsin InhibitorExplored by Combinatorial Library Screening,” J. Biol. Chem.274(30):21037-21043 (1999)). Lysine 15 may be removed for ease ofsynthesis and labeling, and replaced with a less reactive residue. Itcan also be seen that the sequence CLAG (SEQ ID NO: 110) has beenvaried, e.g., CPAG (SEQ ID NO: 111), CQAG (SEQ ID NO: 112), CRAG (SEQ IDNO: 113). These mutations were isolated from the library; however, arethought to have arisen from primer errors, since mutagenesis was notperformed at this amino acid position.

The RGD insert, on either side of the linked Cys residues, comprises thesequence RGD within an 11 amino acid sequence of 11 amino acids replacesthe native sequence, with R at the 4^(th) position. Putting R in the3^(rd) position (EETI #2) was found to decrease binding in the peptidestested. That is, the inserts, which had the sequenceX₁X₂X₃R₄G₅D₆X₇X₈X₉X₁₀X₁₁ (sequence (a)) were superior toX₁X₂R₃G₄D₅X₆X₇X₈X₉X₁₀X₁₁ (sequence (b)), where the subscript indicatesposition in the insert). The length of the loop is based on the distancebetween the adjacent Cys residues, but may be varied between about 9 and13 residues. As shown in FIG. 3, for EETI-II the adjacent Cys residuesare not linked to each other; rather they are linked to other Cysresidues in a peptide “scaffold,” which is a knottin peptide such aslisted above in Tables 1-4.

EETI Sequence Variations

The RGD-containing loop may be varied from the specific sequencesdisclosed. For example the loop sequence of 2.5F, PLPRGDNPPTE (SEQ IDNO: 106) (See below) may be varied in the 8 non-underlined residues to acertain degree without affecting binding specificity and potency. Forexample, if three of the eleven residues were varied, one would haveabout 70% identity to 2.5D. For guidance in selecting which residues tovary, histograms in FIG. 11 present information on likely residues foreach position. For example, in position-3 (the first X, one would mostlikely use a proline residue, based on isolated mutants that hadpositive integrin binding. However, His or Leu are also possiblechoices, as shown by their higher incidence in mutants with goodintegrin binding properties.

The sequences from Table 2 were aligned using NPS@: Network ProteinSequence Analysis, TIBS 2000 March Vol. 25, No. 3 [291]:147-150, CombetC., Blanchet C., Geourjon C. and Deléage G. The alignment was performedat http://npsa-pbil.ibcp.fr/cgi-bin/align_multalin.pl, using defaultparameters. Residues conserved for 90% or more (upper-case letters): 24is 72.73%. The sequences in Table 2 are considered substantiallyidentical to the consensus sequence.

(SEQ ID NO: 53) GCPXGRGDWAPPSCKQDSDCRAGCVCGPNGFCG,where X = any amino acid.

Agouti-Related Protein (AgRP) and Agatoxin Sequences:

The two wild-type proteins AgRP and Agatoxin are quite different insequence, but they have the same three-dimensional fold. As a result,any RGD sequence that works in AgRP will work in Agatoxin, and viceversa.

The following sequences illustrate various RGD mimics, showingimprovements in integrin binding properties obtained by the yeastdisplay molecular evolution process described above. The integrinbinding properties of the peptides were RGD-AgRP #1<#2, <#3:

TABLE 3 AgRP peptides ID no. Sequence SEQ ID NO: RGD-GCVRLHESCLGQQVPCCDPCATCYC RGD CYCRKLGTAMNPCSRT (SEQ ID NO: 54) AgRP#1RGD- GCVRLHESCLGQQVPCCDPCATCYC TGRGDS CYCRKLGTAMNPCSRT (SEQ ID NO: 55)AgRP#2 RGD- CVRLHESCLGQQVPCCDPCATCYC TGRGDSPAS CYCRKLGTAMNPCSRT(SEQ ID NO: 56) AgRP#3 Mini- GCVRLHESCLGQQVPCCDPAATCYC TGRGDSPAS CYCR(SEQ ID NO: 57) RGD-AgRP Mini-RGD- GCIAEDYGKCTWGGTKCCRGRPCRC TGRGDSPASCECT (SEQ ID NO: 58) Agatoxin

A shortened version of AgRP was also prepared. The Mini-AgRP-basedstarting library was:

(SEQ ID NO: 59) GCVRLHESCLGQQVPCCDPAATCYC XXRGDXXXX CYCR.

Variants based on Mini-RGD-AgRP isolated by the techniques describedabove are shown below.

TABLE 4 Mini-RGD-AgRP peptides Number Sequence SEQ ID NO: 3AGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 60) VVRGDWRKR CYCR 3BGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 61) EERGDMLEK CYCR 3CGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 62) ETRGDGKEK CYCR 3DGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 63) QWRGDGDVK CYCR 3EGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 64) SRRGDMRER CYCR 3FGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 65) QYRGDGMKH CYCR 3GGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 66) TGRGDTKVL CYCR 3HGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 67) VERGDMKRR CYCR 3IGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 68) TGRGDVRMN CYCR 3JGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 69) VERGDGMSK CYCR 4AGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 70) RGRGDMRRE CYCR 4BGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 71) EGRGDVKVN CYCR 4CGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 72) VGRGDEKMS CYCR 4DGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 73) VSRGDMRKR CYCR 4EGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 74) ERRGDSVKK CYCR 4FGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 75) EGRGDTRRR CYCR 4GGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 76) EGRGDVVRR CYCR 4HGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 77) KGRGDNKRK CYCR 4IGCVRLHESCLGQQVPCCDPAXTCYC (SEQ ID NO: 78) KGRGDVRRV CYCR 4JGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 79) VGRGDNKVK CYCR 5AGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 80) VGRGDNRLK CYCR 5BGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 81) VERGDGMKK CYCR 5CGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 82) EGRGDMRRR CYCR 5DGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 83) QGRGDGDVK CYCR 5EGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 84) SGRGDNDLV CYCR 5FGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 85) VERGDGMIR CYCR 5GGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 86) SGRGDNDLV CYCR 5HGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 87) EGRGDMKMK CYCR 5IGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 88) IGRGDVRRR CYCR 5JGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 89) EERGDGRKK CYCR 6BGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 90) EGRGDRDMK CYCR 6CGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 91) TGRGDEKLR CYCR 6EGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 92) VERGDGNRR CYCR 6FGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 93) ESRGD VVRKCYCR 7CGCVRLHESCLGQQVPCCDPAATCYC (SEQ ID NO: 94) YGRGDNDLR CYCR

Anti-Angiogenic Activity

The present peptides have been shown to bind to integrins α_(v)β₃andα_(v)β₅, by in vitro experiments in which the peptides were incubatedwith soluble integrins as described above.

Based on present knowledge of cell adhesion and tumorigenesis, it may beexpected that the present peptides will function in vivo as well as invitro, and that they will exhibit anti-angiogenic and anti-proliferativeactivity. It is known that the integrin α_(v)β₃ is required forangiogenesis, see Brooks et al., Science 264:569-571. In order todemonstrate and evaluate such activity, a number of assays, known in theart are included within the present concepts.

Such assays include, but are not limited to, assays of endothelial cellproliferation, endothelial cell migration, cell cycle analysis, andendothelial cell tube formation, detection of apoptosis, e.g., byapoptotic cell morphology or Annexin V-FITC assay, chorioallantoicmembrane (CAM) assay, and inhibition of renal cancer tumor growth innude mice. Examples of such assays are given in U.S. Pat. No. 6,962,974to Kalluri, issued Nov. 8, 2005, entitled “Anti-angiogenic proteins andfragments and methods of use thereof.” For example, C-PAE cells aregrown to confluence in DMEM with 10% fetal calf serum (FCS) and keptcontact inhibited for 48 hours. Control cells are 786-0 (renalcarcinoma) cells, PC-3 cells, HPEC cells, and A-498 (renal carcinoma)cells. Cells are harvested with typsinization (Life Technologies/GibcoBRL, Gaithersburg, Md., USA). A suspension of 12,500 cells in DMEM with1% FCS is added to each well of a 24-well plate coated with 10m/mlfibronectin. The cells are incubated for 24 hours at 37° C. with 5% CO2and 95% humidity. Medium is removed and replaced with DMEM containing0.5% FCS and 3 ng/ml bFGF (R&D Systems, Minneapolis, Minn., USA). Cellsare treated with concentrations of the present engineered peptidesranging from 0.01 to 50m/ml. All wells receive 1μ Curie of ³H-thymidineat the time of treatment. After 24 hours, medium is removed and thewells are washed with PBS. Cells are extracted with 1N NaOH and added toa scintillation vial containing 4 ml of ScintiVerse II (FisherScientific, Pittsburgh, Pa., USA) solution. Thymidine incorporation ismeasured using a scintillation counter. The showing incorporation of³H-thymidine into C-PAE cells treated with varying amounts of thepeptides will show inhibition of cell division.

For animal testing, about two million 786-O cells are injectedsubcutaneously into 7- to 9-week-old male athymic nude mice. In thefirst group of mice, the tumors are allowed to grow to about 700 mm³. Ina second group of mice, the tumors are allowed to group to 100 mm³. Theengineered peptide (e.g., EETI-1.5B, 2.5A, and 2.5D), in sterile PBS isinjected I.P. daily for 10 days, at a concentration of 20 mg/kg for themice with tumors of 700 mm³, and 10 mg/kg for the mice with tumors of100 mm3. Control mice receive either BSA or the PBS vehicle. The resultswill show a change in tumor volume from 700 mm³ for 10 mg/kg peptidetreated, BSA-treated (+), and control mice. Tumors in thepeptide-treated mice will shrink, while tumors in BSA-treated andcontrol mice will grow.

In another known protocol (See again U.S. Pat. No. 6,962,974), about 5million PC-3 cells (human prostate adenocarcinoma cells) are harvestedand injected subcutaneously into 7- to 9-week-old male athymic nudemice. The tumors grow for 10 days, and are then measured with Verniercalipers. The tumor volume is calculated using the standard formula, andanimals are divided into groups of 5-6 mice. Experimental groups areinjected I.P. daily with a test engineered peptide (10 mg/kg/day) or acontrol drug (e.g., an anti-integrin antibody) (10 mg/kg/day). Thecontrol group receives PBS each day. The results will show that anengineered peptide inhibits the growth of tumors as well, or slightlybetter, than did the control drug. The experiment may be repeated atdifferent dosages and times.

EETI, AgRP and Agatoxin Peptide Constructs

The peptides specifically set forth above may be modified in a number ofways. For example, the peptides may be further cross-linked internally,or may be cross linked to each other, or the RGD mimic loops may begrafted onto other cross linked molecular scaffolds. There are a numberof commercially available crosslinking reagents for preparing protein orpeptide bioconjugates. Many of these crosslinkers allow dimeric homo- orheteroconjugation of biological molecules through free amine orsulfhydryl groups in protein side chains. More recently, othercrosslinking methods involving coupling through carbohydrate groups withhydrazide moieties have been developed. These reagents have offeredconvenient, facile, crosslinking strategies for researchers with littleor no chemistry experience in preparing bioconjugates.

The present peptides may be produced by recombinant DNA or may besynthesized in solid phase using a peptide synthesizer, which has beendone for the peptides of all three scaffolds described here. They mayfurther be capped at their N-termini by reaction with fluoresceinisothiocyanate (FITC) or other labels, and, still further, may besynthesized with amino acid residues selected for additionalcrosslinking reactions. TentaGel S RAM Fmoc resin (Advanced ChemTech)may be used to give a C-terminal amide upon cleavage. B-alanine is usedas the N-terminal amino acid to prevent thiazolidone formation andrelease of fluorescein during peptide deprotection (Hermanson, 1996).Peptides are cleaved from the resin and side-chains are deprotected with8% trifluoroacetic acid, 2% triisopropylsilane, 5% dithiothreitol, andthe final product is recovered by ether precipitation. Peptides arepurified by reverse phase HPLC using an acetonitrile gradient in 0.1%trifluoroacetic acid and a C4 or C18 column (Vydac) and verified usingmatrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF) or electrospray ionization-mass spectrosometry(ESI-MS).

When the present peptides are produced by recombinant DNA, expressionvectors encoding the selected peptide are transformed into a suitablehost. The host should be selected to ensure proper peptide folding anddisulfide bond formation as described above. Certain peptides, such asEETI-II can fold properly when expressed in prokaryotic hosts such asbacteria.

Exemplary DNA sequences used for the present peptides are given below:

RGD-EETI#3-based hits (DNA sequences) 1.4B (SEQ ID NO: 95)GGGTGCGTGGGGGGGAGAGGGGATTGGAGCCCGAAGTGGTGCAAACAGGACTCCGACTGCCCGGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 1.5B (SEQ ID NO: 96)GGGTGCACGATCGGGAGAGGGGATTGGGCCCCCTCGGAGTGCAAACAGGACTCCGACTGCCTGGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 1.5F (SEQ ID NO: 97)GGGTGCCACCCGCCGAGAGGGGATAACCCCCCCGTGACTTGCAAACAGGACTCCGACTGCCTGGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.4F (SEQ ID NO: 98)GGGTGCTATCAAGGAAGAGGGGATTGGTCTCCTTCATCGTGCAAACAGGACTCCGACTGCCCAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.5A (SEQ ID NO: 99)GGGTGCCATGTAGGAAGAGGGGATTGGGCTCCTGAAGAGTGCAAACAGGACTCCGACTGCCAAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.5C (SEQ ID NO: 100)GGGTGCGATGGAGGAAGAGGGGATTGGGCTCCTCCAGCGTGCAAACAGGACTCCGACTGCCGAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.5D (SEQ ID NO: 101)GGGTGCCCTCAAGGAAGAGGGGATTGGGCTCCTACATCGTGCAAACAGGACTCCGACTGCCGAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.5F (SEQ ID NO: 102)GGGTGCCCTCGACCAAGAGGGGATAACCCTCCTCTAACGTGCAAACAGGACTCCGACTGCCTAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.5H (SEQ ID NO: 103)GGGTGCCCTCAAGGAAGAGGGGATTGGGCTCCTGAATGGTGCAAACAGGACTCCGACTGCCCAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.5J (SEQ ID NO: 104)GGGTGCCCTCGAGGAAGAGGGGATTGGTCTCCTCCAGCGTGCAAACAGGACTCCGACTGCCAAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA

Dimeric, trimeric, and tetrameric complexes of the present peptides canbe formed through genetic engineering of the above sequences or byreaction of the synthetic cross-linkers with engineered peptidescarrying an introduced cysteine residue, for example on the C-terminusof the peptide. These oligomeric peptide complexes can be purified bygel filtration. Oligomers of the present peptides can be prepared bypreparing vectors encoding multiple peptide sequences end-to-end. Also,multimers may be prepared by complexing the peptides, such as, e.g.,described in U.S. Pat. No. 6,265,539. There, an active HIV peptide isprepared in multimer form by altering the amino-terminal residue of thepeptide so that it is peptide-bonded to a spacer peptide that containsan amino-terminal lysyl residue and one to about five amino acidresidues such as glycyl residues to form a composite polypeptide.Alternatively, each peptide is synthesized to contain a cystine (Cys)residue at each of its amino- and carboxy-termini. The resultingdi-cystine-terminated (di-Cys) peptide is then oxidized to polymerizethe di-Cys peptide monomers into a polymer or cyclic peptide multimer.Multimers may also be prepared by solid phase peptide synthesisutilizing a lysine core matrix. The present peptides may also beprepared as nanoparticles. See, “Multivalent Effects of RGD PeptidesObtained by Nanoparticle Display,” Montet, et al., J. Med. Chem.; 2006;49(20) pp 6087-6093. EETI dimerization may be carried out with thepresent EETI-II peptides according to EETI-II dimerization paper thatjust came out: “Grafting of thrombopoietin-mimetic peptides into cystineknot miniproteins yields high-affinity thrombopoietin antagonist andagonists,” Krause, et al., FEBS Journal; 2006; 274 pp 86-95.

One may also prepare chemically synthesized peptide-based crosslinkingreagents for use in cross-linking the present peptides. The peptide mayfurther contain a fluorescent label (fluorescein) and two or morethiol-reactive maleimide groups introduced at lysine residues spacedalong a flexible backbone composed of glycine, serine, and glutamic acid(Cochran and Stern, 2000; Cochran et al., 2000). The non-repeatingbackbone amino acid sequences are designed to be water-soluble withlittle propensity to form an ordered structure, and to providesufficient length and flexibility to allow integrin binding side chainsto bind simultaneously to a cell surface. Maleimide-to-maleimidedistances for the cross-linkers, in extended conformations for allowingpendant groups to present peptides in the same plane, are approximately45 Angstroms for the dimeric cross-linkers, and 50 Angstroms for thetrimeric, and tetrameric cross linkers, as estimated from molecularmodels.

Other reagents would allow multivalent presentation of integrin bindingpeptides or small protein scaffolds. Ruthenium-based metathesiscatalysts would allow site-specific crosslinking of alkene functionalgroups incorporated into amino acid side chains. The ability tospecifically couple biomolecules using a chemical strategy that does notrely on natural amino acids would be extremely useful in creating smalloligomeric peptide and protein motifs. An example is illustrated in FIG.2. An amphipathic helix is derived from the coiled coil helix of thetranscription factor GCN4, in which hydrophobic positions of heptadrepeat have been exchanged to insert RGD mimics. Further details aregiven in Pack et al., “Tetravalent miniantibodies with high avidityassembling in Escherichia coli.,” J Mol Biol. 1995 Feb. 10;246(1):28-34.

Synergistic sites on fibronectin and other adhesion proteins have beenidentified for enhanced integrin binding (Ruoslahti, 1996; Koivunen etal., 1994; Aota et al., 1994; Healy et al., 1995). The ability toincorporate different integrin-specific motifs into one soluble moleculewould have an important impact on therapeutic development. Crosslinkerswith heterofunctional specificity may be used for creatingintegrin-binding proteins with synergistic binding effects. In addition,these same crosslinkers could easily be used to create bispecifictargeting molecules, or as vehicles for delivery of radionuclides ortoxic agents for imaging and therapeutic applications.

Methods of Use

The present engineered peptides may be used in a variety of ways. If thepeptides are attached to a surface, they may be used to attract/recruitcells to grow on the surface. For example, the present peptides may beapplied to prosthetic devices, implants, bone grafts, and the like topromote tissue growth and healing at the site. They may be attached toculture dishes and promote attachment and differentiation of cells inculture. In addition, the present engineered peptides may be used tomodulate cell binding to selected integrins such as α₅β₁, α_(v)β₃ andα_(v)β₅, particularly α_(v)β₃, by adding the peptides to a cell cultureto prevent cells expressing these integrins from adhering to asubstrate. In a series of experiments using the present RGD-containingpeptides to block adhesion of U87MG glioblastoma cells tovitronectin-coated plates, it was found that the present peptides 2.5F,2.5D, and 1.5D all blocked adhesion better than controls and comparativecompounds FN-RGD and c(RGDyK) (SEQ ID NO: 140) (data not shown). Also,the present 2.5F, 2.5D and 1.5B peptides were tested for blockingadhesion of U87MG glioblastoma cells to fibronectin-coated plates. Inthis case, only echistatin and polypeptide 2.5F blocked adhesion ofU87MG glioblastoma cells to the fibronectin-coated plates. This confirmsthat the RGD-miniprotein 2.5F binds with strong affinity to the α₅β₁integrin subtype.

These binding studies show that the present peptides can be used insoluble form to modulate binding of cells to known cell culturesubstrates (extracellular matrix). Cell binding to integrins can be usedto modulate stem cell self-renewal or differentiation. For example, itis known that stem cells express higher levels of the beta 1-integrinfamily of extracellular matrix receptors than transit amplifying cellsand this can be used to isolate each subpopulation of keratinocyte andto determine its location within the epidermis. See, Watt, “Epidermalstem cells: markers, patterning and the control of stem cell fate,”Philos Trans R Soc Lond B Biol Sci., 1998 Jun. 29; 353(1370): 831-837.Alternatively, the engineered peptides may be prepared and coated onplates or incorporated into polymers or other biomaterials and used as acell culture substrate to promote adhesion by the selected integrin.

The present peptides may also be used to treat proliferative diseases,when administered in soluble form. By attaching to cellular integrins,they block attachment of the cells and inhibit their growth anddevelopment. The present peptides will therefore find use in cancertherapy. The instant peptides are also useful in combination with knownanti-cancer agents. Such known anti-cancer agents include the following:estrogen receptor modulators, androgen receptor modulators, retinoidreceptor modulators, cytotoxic agents, antiproliferative agents,prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIVprotease inhibitors, reverse transcriptase inhibitors, and otherangiogenesis inhibitors. The instant compounds are also useful whencoadminsitered with radiation therapy. The present peptides may also bechemically linked to cytotoxic agents. They may also be used with otherangiogenesis inhibitors. “Angiogenesis inhibitors” refers to compoundsthat inhibit the formation of new blood vessels, regardless ofmechanism. Examples of angiogenesis inhibitors include, but are notlimited to, tyrosine kinase inhibitors, such as inhibitors of thetyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR20),inhibitors of epidermal-derived, fibroblast-derived, or platelet derivedgrowth factors, MMP (matrix metalloprotease) inhibitors, interferon-.a.,interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors,including nonsteroidal anti-inflammatories (NSAIDs) like aspirin andibuprofen as well as selective cyclooxygenase-2 inhibitors likecelecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69,p. 475 (1982); Arch. Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec.,Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin,Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p. 107.(1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol.57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol., Med.,Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)),carboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab.Clin. Med. 105:141 145 (1985)), and antibodies to VEGF (see; NatureBiotechnology, Vol. 17, pp. 963 968 (October 1999); Kim et al., Nature,362, 841 844 (1993).

The present peptides may also be used in vitro as cell labelingreagents, and in vivo as imaging or diagnostic agents, binding to cells,such as tumor cells, which express high levels of a specific integrin.

Synthesis of Soluble Peptides Folding Conditions for EETI-IIPolypeptides

In preparing the present peptides, it is essential that the correctdisulfide linkages be formed, and that the peptide be correctly folded.Glutathione-assisted oxidative folding of the cystine-knot was used. Anexemplary protocol for EETI-II is given below. Large scale foldingreactions were performed with 20% DMSO (v/v) in 0.1 M ammoniumbicarbonate, pH 9 and 2.5 mM reduced glutathione while gently rockingovernight. The final oxidized product was purified by semi-preparativeHPLC using various linear gradients of solvent A and solvent B.Following purification, the peptide was lyophilized and stored untilused. Working concentrations of pure peptide dissolved in purified waterwere determined by amino acid analysis. The purified peptide wasanalyzed by HPLC and ESI-MS.

Solvent (A) is 99.9% water 0.1% TFA, (B) is 10% water 90% MeCN and 0.1%TFA.

Folding Conditions for Mini-AGRP Polypeptides Tris pH 8.0

10 mM reduced glutathione2 mM oxidized glutathione

0.5 M DMSO

with or without 2-4M guanidine (depending on the peptide)1-3 days at room temperature.

Data on synthesized Agouti peptides: The peptides were tested foractivity; the results are as follows, with the peptide designationcorresponding to the sequence given in Table 4: IC50's (obtained bycompeting off binding of ¹²⁵I-echistatin as described above)

WT—1.4±0.7 μM 3F—880±340 nM 6E—130±20 nM 6F—410±80 nM 7C—23±4 nM ImagingProbes

The present polypeptides target α_(v)β₃, α_(v)β₅, and in some cases α₅β₁integrin receptors. They do not bind to other integrins tested. Thus,these engineered integrin-binding polypeptides have broad diagnostic andtherapeutic applications in a variety of human cancers that specificallyoverexpress the above named integrins. As described below, thesepolypeptides bind with high affinity to both detergent-solubilized andtumor cell surface integrin receptors. Furthermore, when used as opticalimaging agents in mouse xenograft models, the tumor/background signalratio generated by these engineered polypeptides is approximately 60%greater than that elicited by an alternative pentapeptide currentlyunder pre-clinical development. Also, the present engineeredhigh-affinity integrin-binding polypeptides can also be labeled withpositron emitting isotopes and changed from optical imaging probes torobust positron emission tomography (PET)-based imaging agents. In aclinical setting, these polypeptides will be used to visualize integrinexpression in the human body for diagnostic and management applicationsin cancer. They can be coupled to radionuclides for therapeuticpurposes. They offer advantages of stability, which reduces toxic uptakeof free radionuclides by the kidneys.

As described above, it is known that the integrin α_(v)β₃ is expressedduring angiogenesis. The α_(v)β₃ (and α_(v)β₅) integrins are also highlyexpressed on many tumor cells including osteosarcomas, neuroblastomas,carcinomas of the lung, breast, prostate, and bladder, glioblastomas,and invasive melanomas The α_(v)β₃ integrin has been shown to beexpressed on tumor cells and/or the vasculature of breast, ovarian,prostate, and colon carcinomas, but not on normal adult tissues or bloodvessels. Therefore, noninvasive methods to visualize and quantifyintegrin expression in vivo are crucial for patient-specific treatmentof cancer with integrin antagonists. Also, the α₅β₁ has been shown to beexpressed on tumor cells and/or the vasculature of breast, ovarian,prostate, and colon carcinomas, but not on normal adult tissue or bloodvessels. The present, small, conformationally-constrained polypeptides(about 33 amino acids) are so constrained by intramolecular bonds, suchas shown in FIG. 3. For example, EETI-II has three disulfide linkages.This will make it more stable in vivo. These peptides target α_(v)integrins alone, or both α_(v) and α₅β₁ integrins. Until now, it isbelieved that the development of a single agent that can bind α_(v)β₃,α_(v)β₅, and α₅β₁ integrins with high affinity and specificity has notbeen achieved. Since all three of these integrins are expressed ontumors and are involved in mediating angiogenesis and metastasis, abroad spectrum targeting agent (i.e., α_(v)β₃, α_(v)β₅, and α₅β₁) willlikely be more effective for diagnostic and therapeutic applications.

The present engineered polypeptides (termed RGD-miniproteins) haveseveral advantages over previously identified integrin-targetingcompounds. They possess a compact, disulfide-bonded core that confersproteolytic resistance and exceptional in vivo stability.

Our studies indicate their half-life in mouse serum to be approximately90 hours (data not shown). Their larger size (˜3-4 kDa) and enhancedaffinity compared to RGD-based cyclic peptides confer enhancedpharmacokinetics and biodistribution for molecular imaging andtherapeutic applications. This is described in connection with FIG. 8.These RGD-miniproteins are small enough to allow for chemical synthesisand site-specific conjugation of imaging probes, radioisotopes, orchemotherapeutic agents. Furthermore, they can easily be chemicallymodified to further improve in vivo properties if necessary. The imagingstudy shown in FIG. 8 shows tumor localization by peptide 2.5D. Thetumor is indicated by an arrow. The near infrared fluorescent Cy5.5label study provides guidance for the preparation of these polypeptidesas ¹⁸F and ⁶⁴Cu-labeled PET imaging probes. In the clinical setting,these imaging agents will play a critical role in identifying patientswhose cancer would benefit most from specific integrin-targeted therapy,and will provide a molecular rationale for why treatments may later failif tumors cease to express these integrins. They may also serve to stagecancer when coupled with existing PET tracers such as2-fluoro-2-deoxy-glucose (FDG). In addition, as described above, theseRGD-miniproteins may be used for treatment of a variety of humancancers, as RGD-based targeting agents have been shown to havetherapeutic efficacy through caspase-mediated apoptosis and cell death(see, Brooks, P. C., Montgomery, A M., Rosenfeld, M., Reisfeld, R A, Hu,T., Klier, G. & Cheresh, D. A. (1994). Integrin alpha v beta 3antagonists promote tumor regression by inducing apoptosis of angiogenicblood vessels. Cell 79, 1157-64.; Chatterjee, S., Brite, K. H. &Matsumura, A (2001). Induction of apoptosis of integrin-expressing humanprostate cancer cells by cyclic Arg-Gly-Asp peptides. Clin Cancer Res 7,3006-11.

Polypeptide synthesis and folding: RGD-miniproteins described below weresynthesized using standard Fmoc-based solid phase peptide synthesis witha CS Bio CS336S automated synthesizer (Menlo Park, Calif.). Thepolypeptides originally contained a lysine at position 15 that wasmutated to a serine to facilitate chemical coupling of imaging probesspecifically to the N-terminus. Crude polypeptide was purified byreversed phase HPLC using a C₁₈ column (Vydac). The correct molecularmass was verified using electrospray mass spectrometry. Polypeptideswere folded with the assistance of dimethyl sulfoxide and glutathione.Folded polypeptides exhibit a distinct chromatographic profile thatallows them to be purified from unfolded or misfolded species byreversed-phase HPLC.

Binding to tumor cells overexpressing α_(v)β₃ integrins: Referring nowto FIG. 9, RGD-miniproteins were tested for their ability to compete forcell surface integrin binding with ¹²⁵I-labeled echistatin, a proteinwhich binds the α_(v)β₃ integrin with a K_(D) of 0.3 nM. U87MGglioblastoma cells, which express ˜10⁵ α_(v)β₃ integrin receptors percell, were used for these studies. We compared the receptor bindingaffinity of loop-grafted FN-RGD (designated FN-RGD), and three of ouraffinity-matured mutants, designated Miniprotein 1.5B, 2.5D, or 2.5F(see Table 2), to that of c(RGDyK) (SEQ ID NO: 140), a pentapeptidecurrently under pre-clinical development for molecular imagingapplications. An EETI-based polypeptide with a scrambled RDG amino acidsequence, designated FN-RDG, served as a negative control. All of theRGD-containing peptides inhibited the binding of ¹²⁵I-labeled echistatinto U87MG cells in a dose dependent manner. Their IC₅₀ values(corresponding to data in FIG. 9) are shown in the Table 5 below.

TABLE 5 IC50 values of 1.5B, 2.5D and 2.5F c(RGDyK) (SEQ ID NO:Loop-grafted Miniprotein Miniprotein Miniprotein Cy5.5 Echistatin 140)FN-RGD 1.5B 2.5D 2.5F IC₅₀ No 4.9 ± 1.0 nM  860 ± 400 nM  370 ± 150 nM 13 ± 3.3 nM  16 ± 6.1 nM  26 ± 5.4 nM IC₅₀ Yes 2.6 ± 0.2 nM 62.9 ± 4.1nM 33.9 ± 13 nM 6.4 ± 3.3 nM 4.2 ± 0.9 nM 3.4 ± 0.8 nM

The above table shows competition binding of engineered peptides with¹²⁵I-echistatin to U87MG tumor cells. Half-maximal inhibitoryconcentrations (IC₅₀) represent the standard deviation of data measuredon at least three separate days. Data for unlabeled and Cy5.5-labeledpeptides are shown. Site-specific labeling of RGD miniproteins with anear-infrared optical imaging probe: The free N-terminal amine of ourpolypeptides was used for site-specific attachment of Cy5.5, a nearinfrared imaging probe.

Our evolved mutants were shown to bind to U87MG cells with a 50 to80-fold higher affinity than both of the parental loop-grafted FN-RGDand c(RGDyK) (SEQ ID NO: 140).

Unique integrin binding specificities: Since U87MG cells have been shownto express α_(v)β₃, α_(v)β₅, and α₅β₁ integrins, it was necessary to useanother means to measure integrin-binding specificity. This was done bycompetition of ¹²⁵I-echistatin to detergent-solubilized integrinreceptors coated onto microtiter plates (see FIG. 10). As expected,echistatin binds strongly to all of the tested integrins. The scrambledFN-RDG miniprotein, the negative control, did not bind to any of theintegrins used in this study. All peptides bound to α_(v)β₃ and α_(v)β₅integrins to some degree, with the engineered RGD-miniproteins 1.5B,2.5D, and 2.5F showing the strongest levels of binding. This isconsistent with previous studies which have shown α_(v) integrinreceptors can accommodate a wide range of RGD-containing cyclicstructures. Interestingly, the RGD-miniprotein 2.5F binds with strongaffinity to the α₅β₁ integrin subtype, while RGD-miniproteins 1.5B and2.5D exhibit only minimal binding to this receptor. However, since α₅β₁integrins are expressed on many tumors and are all involved in mediatingangiogenesis and metastasis, a broad spectrum agent that targets allthree integrins will be useful for diagnostic and therapeuticapplications. With the exception of echistatin, all of theRGD-containing peptides bound weakly to the α_(iib)β₃ receptor, showingthe specificity of the present peptides for the αv and α5-containingintegrin heterodimers. This characteristic is valuable for molecularimaging and therapeutic applications, since binding to a_(iib)β₃ onplatelet cells prevents blood clotting and would lead to non-specific invivo effects.

(SEQ ID NO: 28) 1.5B: GC TIGRGDWAPSE CKQDSDCLAGCVCGPNGFCG(SEQ ID NO: 49) 2.5 D GC PQGRGDWAPTS CKQDSDCRAGCVCGPNGFCG(SEQ ID NO: 50) 2.5F GC PRPRGDNPPLT CKQDSDCLAGCVCGPNGFCG

Viewing 1.5B and 2.5D together, one observes the identical “GRGDWAP”(SEQ ID NO: 108) motif that these peptides have in common, and would beexpected to confer the integrin specificity observed. On the other hand,the unique specificity of 2.5F (strong affinity to the α₅β₁ integrinsubtype), is reflected in the additional proline residues.

The proline rich sequence of 2.5F may constrain the binding epitope intoa favorable conformation for these high affinity interactions. We arenow pursuing molecular modeling and structural studies to provideinsight into the unique binding integrin specificity of this peptide.

In another experiment, the ability of engineered knottin peptides toblock U87MG cell adhesion to vitronectin- and fibronectin-coatedmicrotiter plates. Vitronectin is a natural ligand for severalintegrins, including α_(v)β₃ and α_(v)β₅. The RGD-containing peptideswere all able to inhibit U87MG cell adhesion to vitronectin-coatedplates in a dose-responsive manner. The IC₅₀ values of cell adhesioncorrelated with the ¹²⁵I-echistatin competition binding data (above),indicating that inhibition of cell adhesion is directly related tointegrin binding events. Fibronectin also binds to several integrins,(including α_(v)β₃ and α₅β₁), but the α₅β₁ integrin receptor isgenerally selective for fibronectin. We found that only echistatin andknottin peptide 2.5F were able to block U87MG cell adhesion tofibronectin-coated plates, consistent with their ability to bind bothα_(v) and α₅β₁ integrins with high affinity. The FN-RDG2 negativecontrol was not able to inhibit U87MG cell adhesion to vitronectin orfibronectin, as expected.

Engineered Knottin Peptides Exhibit High Stability in Serum

The stability of the knottin peptide 2.5D upon exposure to human ormouse serum at 37° C. was measured. Reversed-phase HPLC was used toquantify the amount of intact knottin peptide remaining at various timespost incubation. We found that approximately 90% of the peptide remainedafter incubation for 24 h in human and mouse serum, with approximately70% remaining after 96 h.

Soluble Expression of Engineered AgRP Peptides

P. pastoris was chosen for recombinant expression of the engineered AgRPpeptides, as it has been successfully used to express proteins withdisulfide bonds and significant secondary structure. The eukaryoticquality control machinery in the secretory pathway of yeast should helpensure proper folding and high levels of soluble expression of the AgRPpeptides, which have four disulfide bonds and complex folds. Usingconditions and procedures described in the P. pastoris expression kit(Invitrogen K1750-01), AgRP clones 6C, 7A (named above as 5E), 7C, 7E,and 7J (named above as 6B) were produced in yields of 3-10 mg/L culture.

TABLE 5 Sequences of Additional AgRP mutants isolatedfrom flow cytometry sort rounds 6 and 7. Clone Loop 4 sequence 7A (5E)GCVRLHESCLGQQVPCCDPAATCYCSGRGDND (SEQ ID NO: 114) LVCYCR 7BGCVRLHESCLGQQVPCCDPAATCYCKGRGDAR (SEQ ID NO: 115) LQCYCR 7EGCVRLHESCLGQQVPCCDPAATCYCVGRGDDN (SEQ ID NO: 116) LKCYCR 7J (6B)GCVRLHESCLGQQVPCCDPAATCYCEGRGDRD (SEQ ID NO: 90) MKCYCR

The engineered AgRP peptides were expressed with N-terminal FLAG epitopetags (DYKDDDDK) SEQ ID NO: 138 and C-terminal hexahistidine tags for useas handles in purification and cell binding assays through antibodydetection. The expressed peptides were purified by Ni-affinitychromatography and determined to be >90% pure by reversed-phase HPLC andgel-filtration chromatography. SDS-PAGE analysis was performed onreduced and non-reduced. Peptide composition was confirmed and exactconcentrations were determined by amino acid analysis (data not shown),and masses were obtained by MALDI-TOF mass spectrometry.

To determine whether the FLAG and hexahistidine tags would interferewith α_(v)β₃ integrin binding, one of the AgRP clones, 7C, was preparedwithout epitope tags by solid-phase peptide synthesis using standardFmoc chemistry. The crude, reduced peptide was purified byreversed-phase HPLC, then oxidized with glutathione and DMSO, aspreviously described. The fully oxidized peptide was purified fromunfolded and misfolded states by reversed-phase HPLC, and the mass wasconfirmed by mass spectrometry.

To compare the α_(v)β₃ integrin binding affinities of synthetic andrecombinant AgRP peptides, a competition binding assay using U87MGglioblastoma cells, which express approximately 10⁵ α_(v)β₃ receptorsper cell, as well as αvβ5 and α5β1 integrins. Recombinant AgRP peptide7C (20 nM) was pre-incubated with 10⁵ cells and binding was thencompeted off using synthetic AgRP peptide 7C at concentrations rangingfrom 1 nM to 500 nM. After washing, the cells were stained with afluorescein-conjugated anti-FLAG antibody and then analyzed by flowcytometry. Competition of synthetic peptide by recombinant peptide wasperformed analogously. These experiments gave essentially identicalhalf-maximal inhibitory concentration (IC₅₀) values (22±3 nM and 23±6nM, respectively; suggesting that both recombinant and synthetic AgRPpeptides are correctly folded and that the FLAG and His epitope tags onthe recombinant peptide do not interfere with integrin binding.

Integrin Binding Affinity and Specificity of Engineered AgRP Peptides.

Direct equilibrium binding titrations of the recombinant engineered AgRPpeptides were performed on U87MG glioblastoma cells. Peptides wereincubated with cells for 3 h at 4° C., followed by staining with afluorescein-conjugated anti-His antibody and analysis by flow cytometry.Equilibrium binding constant (K_(D)) values were obtained by fittingplots of concentration versus mean fluorescence intensity to a sigmoidalcurve using KaleidaGraph software.

TABLE 6 Binding and cell adhesion data summary for engineered AgRPpeptides. binding (K_(D); nM) adhesion (IC₅₀; nM) Clone U87MG K562-αvβ3K562-αvβ3 6C 13 ± 2  15 ± 4  650 ± 250 7A 0.78 ± 0.37 0.89 ± 0.36 61 ±19 7C 1.8 ± 0.6 2.4 ± 0.8 12 ± 6  7E 1.4 ± 0.5 1.6 ± 1.1 9.9 ± 5.8 7J8.3 ± 1.8 16 ± 8  200 ± 150 Echistatin nd nd 3.2 ± 2.7 nd = notdetermined

As shown above, all five engineered AgRP peptides tested bound with lownanomolar to high picomolar affinity, and the tightest binder, 7A,isolated from sort round 7, showed 17-fold improvement over the worstbinder, 6C, isolated from sort round 6. The saturation levels for thedifferent clones vary roughly with affinity. This suggests that themutants have different binding off-rates that dictate their K_(D)values, with weaker binding clones having faster off-rates.Alternatively, the differences in saturation levels could be due tointegrin receptor clustering that is differentially elicited orstabilized by clones with varying affinities.

To determine the binding specificities of the AgRP peptides for α_(v)β₃integrin versus the αvβ5 and α5β1 integrins also expressed on U87MGcells, K562 leukemia cells that had been stably transfected withindividual α and β integrin subunits were used. The engineered AgRPpeptides were tested for binding to untransfected K562 cells, whichintrinsically express α5β1 integrin. Equilibrium binding assays wereperformed on the untransfected K562 cells as described above for theU87MG cells. Three peptide concentrations were tested. Negligible signalover background levels (cells stained with fluorescein-conjugatedanti-His antibody alone) even at 500 nM, the highest concentrationtested, was observed. This demonstrated that the engineered AgRPpeptides do not appreciably bind to α5β1 integrin, and that the K562cells transfected to express other integrins would be useful indetermining integrin-binding specificity.

The engineered AgRP peptides were tested for binding to K562 cellsexpressing αvβ5 αiiiβ3 or αvβ3 integrins. The peptides were tested atthree concentrations with very little signal over background for cellsexpressing αvβ5 integrin, even at 500 nM, the highest concentration. Thepeptides bound weakly to the K562 cells expressing αiiβ3 integrins and,as expected, strongly to the K562 cells expressing αvβ3 integrins. Inorder to determine K_(D) values for the engineered AgRP peptides againstK562-αiiβ3 and K562-αvβ3 cells, binding titrations were done over alarger range of concentrations. K_(D) values for AgRP peptide binding toK562-αvβ3 cells were essentially identical to the K_(D) values obtainedfor the U87MG cells, indicating that binding of engineered AgRP peptidesto U87MG cells is mediated by αvβ3 integrin (Table 7). K_(D) values forpeptide binding to K562-αiibβ3 cells could not be determined becausebinding was still increasing at the highest concentration (5 μM) ofpeptides tested. However, from the data, it may be estimated that theK_(D) values for AgRP peptide binding to the K562-αiibβ3 cells are muchgreater than 100 nM.

Inhibition of Vitronectin-Mediated Cell Adhesion by Engineered AgRPPeptides.

To determine whether the engineered AgRP peptides could inhibit celladhesion mediated by vitronectin, the primary ligand for α_(v)β₃integrin, K562-α_(v)β₃ cells were incubated with varying concentrationsof peptides in microtiter wells coated with vitronectin to determine theability of the peptides to inhibit cell adhesion. The engineered AgRPpeptides were able to block vitronectin-mediated adhesion of theK562-α_(v)β₃ cells with IC₅₀ values ranging from 9.9 to 650 nM (Table7). The IC₅₀ values for inhibition of cell adhesion were 6- to 67-foldgreater than the K_(D) values against the K562-α_(v)β₃ cells. Thisdifference may be a result of multivalent interactions between the cellsurface α_(v)β₃ integrins and the immobilized vitronectin, therebymaking it more difficult for the peptides to compete.

Whether the engineered AgRP peptides could block vitronectin-mediatedadhesion to the U87MG cells was tested using an analogous assay.However, adhesion of the U87MG cells was only partially blocked by thepeptides, even at concentrations up to 1 μM. In contrast, theRGD-containing disintegrin echistatin, which binds strongly to α_(v)β₃,αvβ5, α5β1, and αiibβ₃ integrins, blocked U87MG cell adhesion tovitronectin with an IC₅₀ of 5.8 nM. The AgRP peptides may noteffectively block U87MG adhesion compared to the K562-α_(v)β₃ cellsbecause the αvβ5 integrins co-expressed on the surface of the U87MGcells could also contribute to vitronectin-mediated adhesion andcompensate for the loss of α_(v)β₃ integrin function. These data providefurther evidence that the engineered AgRP peptides bind to α_(v)β₃ butnot to αvβ5 integrins.

Mutagenesis of AgRP Loops 1, 2 and 3.

Modification of the remaining AgRP loops (loops 1, 2, or 3) were studiedfor their effect on integrin binding affinity or specificity, and/or onthe tolerance of the other AgRP loops to mutagenesis for further proteinengineering studies. This is illustrated as follows:

(SEQ ID NO: 117) GCXXXXXXXCLGQQVPCCDPAATCYCYGRGDNDLRCYCR

In this sequence loops one and four are underlined.

(SEQ ID NO: 118) GCVRLHESCXXXXXXXCCDPAATCYCYGRGDNDLRCYCR

In this sequence loops two and four are underlined.

(SEQ ID NO: 119) GCVRLHESCLGQQVPCCXXXXXXCYCYGRGDNDLRCYCR

In this sequence loops three and four are underlined. Loop 4 containsthe engineered regions previously described, and loops 1-3 are beingmodified in this example.

Yeast-displayed libraries using clone 7C as a starting point, with loops1, 2, or 3 as shown above individually substituted with randomizedsequences using degenerate codons were prepared. These randomized looplibraries were subjected to four rounds of screening by FACS toascertain whether it would be possible to select mutants that retainedbinding to α_(v)β₃ integrin. In each screening round, the yeast werelabeled for peptide expression using the cMyc epitope and incubated with50 nM α_(v)β₃ integrin, followed by staining with fluorescently labeledsecondary antibodies. Although the initial libraries showedsignificantly diminished binding to α_(v)β₃ integrin compared to theparent clone 7C, mutants that retained affinity for α_(v)β₃ integrinwere enriched after four rounds of screening (data not shown).

After sort round four, six yeast-displayed clones were chosen at randomfrom each loop-mutagenized library (Table 8), and were tested for theirability to bind integrins.

TABLE 7 Modified Loop Sequences of AgRP Loops 1, 2 and 3 Clone^(a)Modified Loop Sequence 1-1 ASGSGDP SEQ ID NO: 120 1-2RPLGDAG SEQ ID NO: 121 1-3 LAGLSGP SEQ ID NO: 122 1-4RSASVGG SEQ ID NO: 123 1-5 IASGLFG SEQ ID NO: 124 1-6DLYGSHD SEQ ID NO: 125 2-1 GGSVGVE SEQ ID NO: 126 2-2DPRVGVR SEQ ID NO: 127 2-3 ADTLMAA SEQ ID NO: 128 2-4EWGRGGD SEQ ID NO: 129 2-5 GSWGTLA SEQ ID NO: 130 2-6WGSILGH SEQ ID NO: 131 3-1 GTPKPE SEQ ID NO: 132 3-2SRSDAH SEQ ID NO: 133 3-3 SGLGNR SEQ ID NO: 134 3-4QGREQS SEQ ID NO: 135 3-5 TVTNSR SEQ ID NO: 136 3-6TSKQHH SEQ ID NO: 137 ^(a)First number indicates mutated loop, secondnumber indicates clone number.

Yeast displaying each mutant were treated with 50 nM αvβ3, 50 nM αiibβ3, or 50 nM αv β5 integrin, then stained with an appropriatefluorescein-conjugated anti-integrin antibody and analyzed by flowcytometry. None of the clones bound to αv β5 integrin (data not shown).In contrast, all of the clones bound αvβ3 integrin at levels close tothat of the parent clone 7C. The clones also weakly bound to αiib β3integrin, albeit at lower levels compared to the parent clone 7C. Theratio of αv β3 binding to αiib β3 binding was increased for all of theclones over the parent clone 7C. These data suggest that AgRP loops 1,2, and 3 are in principle tolerant to mutagenesis and they maycontribute to binding specificity through either direct integrincontacts or through structural changes in the engineered AgRP peptides.

The above binding studies demonstrate that the engineered AgRP peptidesare selective for αv β3 over several other integrins that also recognizeRGD sequences, namely αv β5, α β1, and αiib β3. The engineered peptidesdid not bind at all to αv β5 or α₅ β1, whereas weak binding was observedagainst αiib β3 integrin. It has been a challenge in the past to selectpeptides that are selective for αv β3 over αiib β3 and vice versa.Without wishing to be bound by any theory, it is believed that theexamples here provide guidance for design of such selective peptides.RGD ligands bind to αv β3 and αiib β3 near β-propeller loops of the asubunit that form a cap subdomain and a so-calledspecificity-determining loop in the β3 subunit. Structural differencesbetween αv and αiib are responsible for variations in the cap subdomainand the β3 specificity-determining loop, while the remainder of the αvand αiib β-propeller structures are conserved. Consequently, specificityof RGD-containing peptides for αv β3 versus αiib β3 is controlled by thedeeper β-propeller pocket of αiib. The Arg residue in RGD must be in anextended conformation to reach into the αiib pocket to hydrogen bondwith αiib-Asp224, while αv-Asp150 and αv-Asp218 residues are found in ashallower pocket. Furthermore, αiib β3 shows a preference for aliphaticresidues flanking RGD, as αv-Asp218 is replaced by a hydrophobic Phe231in αiib. It is postulated that the engineered AgRP peptides tested herehave predominantly hydrophilic or charged residues flanking RGD, whichmay clash with αiib-Phe231.

Mutagenesis and Screening of AgRP Loop 1, 2, and 3 Libraries

Libraries of AgRP clone 7C (Table 8) with loop 1, 2, or 3 substitutedwith 7, 7, or 6 randomized amino acid residues, respectively, wereprepared as described above. For screening, each library was incubatedwith 50 nM αv β3 integrin and 2×10⁷ cells were sorted by FACS asdescribed above. Three additional rounds of FACS were performed toenrich for a pool of clones that retained binding to αv β3 integrin. Inthese subsequent sort rounds the libraries were over sampled at least10-fold to ensure diversity was maintained, but the integrinconcentration was kept at 50 nM. Clones from the fourth round of sortingwere isolated and sequenced as described above.

Recombinant and Synthetic Production of Engineered AgRP Mutants

Peptides were expressed recombinantly using the Multi-Copy PichiaExpression Kit (Invitrogen K1750-01). The open reading frame encodingthe clone of interest was inserted into pPIC9K plasmid between the AvrIIand MluI restriction sites. In addition, DNA encoding for a FLAG tag wasinserted between SnaBI and AvrII sites, while DNA encoding for ahexahistidine tag was inserted between MluI and NotI restriction sites.˜10 μg of plasmid was linearized by cutting with SacI thenelectroporated into the P. pastoris strain GS115. Yeast were allowed torecover on RDB plates and were then transferred to YPD plates containing4 mg/mL geneticin. Geneticin-resistant colonies were grown in BMGY andthen induced in BMMY. Cultures were grown for 3 days with methanolconcentration maintained at ˜0.5%.

AgRP Clone 7C was also prepared without FLAG or His tags usingsolid-phase peptide synthesis on a CS Bio peptide synthesizer (MenloPark, Calif.) using standard Fmoc chemistry. The peptide was purified byreversed-phase HPLC and then folded using 4 M guanidine, 10 mM reducedglutathione, 2 mM oxidized glutathione, and 0.5 M DMSO at pH 7.5. Thecorrectly folded peptide was separated from unfolded and partly foldedpeptides by reversed-phase HPLC, where it appeared as a single peak witha shorter retention time than unfolded or misfolded precursors. Allpeptides, recombinant and synthetic, were characterized by amino acidanalysis (AAA Service Laboratory, Damascus, Oreg.) and MALDI-TOF massspectrometry (Stanford Protein and Nucleic Acid Facility), which gave asingle peak corresponding to the fully folded protein containing fourdisulfide bonds.

Cell Binding Assays

All cell lines were cultured at 37° C. with 5% CO2. Adherent U87MG cellswere obtained from ATCC and cultured in DMEM media (Gibco 11995)supplemented with 10% fetal bovine serum. Untransfected K562 cells (α5β1-positive) were obtained from ATCC and cultured in suspension in IMDMmedia (Gibco 12440) supplemented with 10% fetal bovine serum. K562 cellsstably transfected with αv β3, αv β5, or αiib β3 integrins were obtainedfrom S. Blystone (Blystone, S. D., Graham, I. L., Lindberg, F. P. &Brown, E. J. (1994). Integrin avb3 differentially regulates adhesive andphagocytic functions of the fibronectin receptor α5b1. J. Cell Biol.127, 1129-1137) and were grown in media supplemented with 10 μg/mLgeneticin. Equilibrium binding assays were performed with 10⁵ cells perreaction. Cells were suspended in IBB with varying amounts of engineeredAgRP peptide at 4° C. for 3 h with gentle rocking. The cells were washedand resuspended in BPBS with a 1:40 dilution of fluorescein-conjugatedanti-6X-His antibody (Bethyl A190-113F) and incubated on ice for 20 min.After washing, the cells were analyzed by flow cytometry using a BDFACSCalibur instrument and CellQuest software (Becton Dickinson,Franklin Lakes, N.J.). Mean fluorescence intensity values for each cellpopulation was plotted against concentration on a log scale. Data wasfit to sigmoidal curves to obtain equilibrium dissociation constantsusing KaleidaGraph (Synergy Software), and is presented as averagevalues with standard deviations. Each assay was performed a minimum ofthree times.

Modifications of EETI-II Loops 2 and 3

This example involves the creation of EETI loop-substituted libraries inwhich a single cysteine-flanked loop of EETI (loop 2 or loop 3) wassubstituted with randomized amino acid sequences of variegated lengthsin order to explore the tolerance of the EETI scaffold for differentloop sizes and amino acid compositions. Libraries were generated byoverlap extension PCR using degenerate NNS oligonucleotides (N=anynucleotide, S=G or C). Six libraries in total were generated: twolibraries of EETI loop 2 variants with substitution lengths of 7 aminoacids (EL2-7) and 9 amino acids (EL2-9), and four libraries of EETI loop3 variants with substitution lengths of 6 amino acids (EL3-6), 7 aminoacids (EL3-7), 8 amino acids (EL3-8), and 9 amino acids (EL3-9). EETIloop 1, which is responsible for binding to trypsin, was used as ahandle to probe the structural integrity of the EETI loop-substitutedclones. Library DNA was electroporated into the S. cerevisiae EBY100strain with linearized yeast-display plasmid as previously described. Byperforming dilution plating, it was estimated that the sizes of theloop-substituted libraries ranged from 5×10⁶-1×10⁷ transformants. Atleast 50 clones from each of the six original libraries were sequencedto confirm that the substituted loops were of the correct lengths andhad diverse amino acid compositions. The amino acid frequencies of theloop-substituted regions were similar to those expected for a degenerateNNS codon library.

Isolation of EETI Loop-Substituted Trypsin-Binding Clones

It has been previously demonstrated that retention of the correctpairings of disulfide-bonded cystines in EETI can be examined by testingfor trypsin binding. Each of the EETI loop-substituted libraries wasscreened for clones that were both displayed on the yeast cell surface(as detected by indirect immunofluorescence against the C-terminal cMycepitope tag) and properly folded (as determined by their ability to bindfluorescently-labeled trypsin) using dual-color fluorescence-activatedcell sorting (FACS). By performing repeated rounds of FACS on eachyeast-displayed library, each time collecting the top 1-2% oftrypsin-binding clones, EETI loop-substituted clones that retained thehighest levels of trypsin-binding ability were enriched. After fourrounds of sorting, a pool of clones that showed moderate to wild-typelevels of trypsin binding had been isolated from each library.

The amino acid frequencies of the enriched library populations alsodiffered from their original, unsorted counterparts. Notably, glycinewas enriched in all EETI loop-substituted libraries compared to thestarting libraries, and cysteine virtually disappeared from alltrypsin-binding clones, except in the EL3-7 library. Apart from glycine,hydrophilic residues predominated in the loop-substituted positions ofenriched clones, which was expected given their solvent-accessibility.EETI loop 2-substituted clones were relatively tolerant of diversityacross all loop positions. Glycine comprised approximately 25-30% of allamino acids in EETI loop 2-substituted trypsin-binding clones at allpositions in the loop except the second. On average, EETI loop2-substituted sequences of both 7- and 9-amino acids containedapproximately 2 glycine residues per clone. Proline and serine, residuesthat commonly populate turn segments, predominated in the secondposition of EETI loop 2-substituted variants (FIG. 5).

The overall diversities of EETI loop 3-substituted clones were slightlyhigher than those of loop 2-substituted clones. The greatest levels ofdiversity occurred in the middle positions of the substituted loops ofloop 3 variants while the first, penultimate, and final positions hadthe lowest diversities. Approximately 75% of all EL3-9 clones began withone of four preferred residues: asparagine, arginine, valine, andhistidine. The most common amino acids for the penultimate and finalpositions of EETI loop 3 were glycine and tyrosine, respectively; nearlya quarter of all loop 3 substituted sequences from enriched clones endedin a glycine-tyrosine doublet. We observed the aforementioned trends intolerated EETI loop 3 substituted clones across all loop lengths.

EETI loop 3 was very tolerant of substitution with 6, 8, and 9 aminoacid sequences, but surprisingly did not appear to be tolerant of a7-amino acid loop.

The Table 9 below show frequencies of amino acid substitutions indifferent positions, taken from the enriched library:

TABLE 8 Amino Acid Substitutions EETI Loop 3 Substituted with 9 AminoAcids Loop Position 1 N (36%) R (15%) V (15%) 2 R (19%) K (15%) T (13%)N (10%) P (10%) 3 N (21%) T (21%) R (11%) 4 N (17%) T (17%) R (15%) 5 R(19%) G (13%) N (12%) H (10%) 6 G (17%) N (13%) T (13%) R (12%) D (10%)7 P (12%) T (12%) L (10%) M (10%) R (10%) 8 G (60%) S (17%) 9 Y (85%)EETI Loop 3 Substituted with 8 Amino Acids Loop Position 1 R (27%) I(20%) V (12%) N (11%) L (9%) 2 H (16%) R (12%) N (11%) G (9%) K (9%) S(9%) 3 S (18%) N (16%) R (12%) T (12%) 4 G (20%) R (18%) K (12%) T (9%)5 R (14%) H (11%) G (11%) Q (11%) 6 R (16%) G (14%) H (9%) 7 G (38%) S(14%) A (11%) R (11%) K (9%) 8 Y (50%) F (14%) T (11%) EETI Loop 3Substituted with 7 Amino Acids Loop Position 1 R (18%) N (17%) D (11%) L(11%) 1 (9%) S (9%) 2 R (14%) S (12%) G (11%) L (11%) P (11%) T (9%) 3 G(18%) S (16%) N (11%) P (11%) R (9%) T (9%) 4 P (12%) T (12%) S (9%) Y(9%) 5 C (14%) G (14%) P (12%) R (9%) T (9%) 6 G (36%) R (20%) A (9%) 7Y (38%) F (20%) C (18%) EETI Loop 3 Substituted with 6 Amino Acids LoopPosition 1 D (27%) N (27%) H (18%) 2 T (21%) K (10%) P (10%) Q (10%) R(10%) S (10%) 3 R (24%) D (10%) L (10%) 4 S (34%) T (14%) D (10%) 5 G(34%) R (16%) N (14%) 6 Y (31%) T (23%) EETI Loop 2 Substituted with 7Amino Acids Loop Position 1 G (32%) S (19%) V (9%) 2 P (21%) S (19%) V(9%) 3 G (26%) S (13%) V (9%) 4 G (24%) L (11%) A (9%) 5 G (43%) S (13%)6 G (21%) S (21%) L (13%) V (9%) 7 G (19%) S (15%) R (13%) V (11%) L(9%) 8 S (23%) G (17%) R (11%) 9 G (38%) P (13%) A (9%) Q (9%) S (9%)EETI Loop 2 Substituted with 9 Amino Acids Loop Position 1 G (26%) D(18%) V (11%) N (10%) 2 P (44%) G (21%) 3 G (41%) A (10%) 4 G (21%) L(13%) V (11%) A (10%) 5 G (33%) S (15%) R (10%) 6 G (38%) S (13%) 7 G(44%) T (11%) E (10%)

Imaging

Site-specific labeling with imaging probe: The free N-terminal amine ofthe engineered peptide was used for site-specific attachment of Cy5.5, anear-infrared imaging probe. Cy5.5 monofunctional N-hydroxysuccinimideester (Amersham Biosciences) was covalently-coupled to all of thepolypeptides described above, and the complexes were purified byreversed-phase HPLC. The molecular masses of the conjugated polypeptideswere confirmed by mass spectrometry (data not shown). Interestingly,Cy5.5 conjugation slightly increased the affinity of the polypeptides toU87MG cells; however, the Cy5.5-labeled FN-RDG negative controlexhibited no binding.

In Vivo Optical Imaging of Tumors in Mouse U87MG Xenograft Models

Whole-body imaging of subcutaneous mouse xenografts were imaged with theIVIS 200 system (Xenogen) and quantified with Living Image 2.50.1software. FIG. 8A shows typical NIR fluorescent images of athymic nudemice bearing subcutaneous U87MG glioblastoma tumors after intravenous(iv) injection of 1.5 nmol of Cy5.5-labeled RGD-miniprotein 2.5D, or theCy5.5-labeled FN-RDG negative control. The fluorescence intensity of thetumor to normal tissue (T/N) ratio as a function of time is depicted inFIG. 8B, which also includes the corresponding values for iv injectionof Cy5.5-labeled loop grafted FN-RGD and c(RGDyK) (SEQ ID NO: 140). TheCy5.5-labeled RGD-miniprotein 2.5D shows approximately a 60% greater T/Nratio at both early and late time points compared to both the FN-RGD andthe c(RGDyK) (SEQ ID NO: 140) pentapeptide. These results indicate thatintegrin binding affinity plays a role in tumor targeting, and providesa strong foundation for clinical translation of RGD-miniproteins as ¹⁸Fand ⁶⁴Cu-labeled PET imaging probes.

Preparation of Radiolabeled Integrin-Binding Polypeptides for microPETImaging.

Integrin binding polypeptides are conjugated to ¹⁸F and ⁶⁴Cu radioprobesfor microPET imaging, which is PET based imaging in small animals. Bothradioprobes are studied due to potential differences in metabolism,pharmacokinetics, and biodistribution.

Polypeptide Synthesis: Polypeptides are synthesized, folded, purified,and characterized as described above.

Preparation of 4-[¹⁸F] fluorobenzoyl-labeled polvpeptides ([¹⁸F] FB):[¹⁸] FB-labeled polypeptides are prepared by conjugation of theN-terminal amine with N-succinimidyl 4-[¹⁸F]fluorbenzoate ([¹⁸F]SFB)under slightly basic conditions as previously described. (See, Chen, X.,Liu, S., Hou, Y., Tohme, M., Park, R, Bading, J. R & Conti, P. S.(2004). MicroPET imaging of breast cancer alpha v-integrin expressionwith Cu-labeled dimeric RGD peptides. Mol Imaging Bioi 6, 350-9.; andChen, X., Park, R, Tohme, M., Shahinian, A H., Bading, J. R & Conti, P.S. (2004). MicroPET and autoradiographic imaging of breast cancer alphav-integrin expression using ¹⁸F- and ⁶⁴Cu labeled RGD peptide. BioconjugChem 15, 41-9).

Briefly, [¹⁸F]SFB are purified by semipreparative HPLC, and theappropriate fraction are collected, diluted with water and trapped by aC-18 cartridge. The cartridge will then be washed with water and blowndried with Argon. [¹⁸F]SFB is eluted with acetonitrile and rotovapped todryness. The dried [¹⁸F]SFB is dissolved in dimethyl sulfoxide andallowed to react with polypeptides in sodium phosphate buffer. Finalpurification is done by semipreparative HPLC. (See, Zhang, X., Xiong,Z., Wu, Y., Cai, W., Tseng, J. R, Gambhir, S. S. & Chen, X. (2006).Quantitative PET imaging of tumor integrin alphavbeta3 expression with18F-FRGD2. J Nucl Med 47, 113-21.)

Before ¹⁸F is used, synthesis and purification conditions should befirst validated with nonradioactive ¹⁹F-labeled polypeptides, andconfirmed using mass spectrometry.

Preparation of DOTA-conjugated polypeptides: 1,4,7,10-tetradodecane-N,N′, N″, N′″-tetraacetic acid (DOTA) are conjugated to polypeptides in amanner similar to that described before. (See, Cheng, Z., Xiong, Z.,Subbarayan, M., Chen, X. & Gambhir, S. S. (2007).) Briefly, DOTA isactivated with 1-ethyl-3-[3-(dimethylamino)propyl]carboiimide at pH 5.5for 30 minutes (4° C.) with a molar ratio ofDOTA:EDC:N-hydroxysulfonosuccinimide=1:1:0.8. Polypeptides are then beadded to the prepared sulfosuccinimidyl ester of DOTA in a stoichiometryof 5:1. The reaction is mixed at pH 8.5-9.0 overnight (4° C.). Theresulting DOTA-conjugated polypeptides are then purified by reversedphase HPLC on a semipreparative C-18 column, and stored as a lyophilizedsolid. The mass is verified by electrospray mass spectrometry.

Preparation of [⁶⁴Cu]-DOTA-polypeptide radiotracers: The DOTA-conjugatedpolypeptides are radiolabeled with ⁶⁴Cu by incubation of 5 mCi ⁶⁴CuCl₂in 0.1 N NaOAc, pH 5.5 for 1 h at 50° C., and terminated withtrifluoroacetic acid. The radiolabeled complex is then be purified byHPLC, dried by rotovap, reconstituted in phosphate buffered saline andpassed through a 0.22 11 m filter for animal experiments. Before ⁶⁴Cu isused, synthesis and purification conditions should be first validatedwith nonradioactive “mock” Cu-DOTA-conjugated polypeptides, andconfirmed using mass spectrometry.

In-Vitro Characterization of Radiolabeled Integrin-Binding Polypeptidesfor microPET Imaging

Nonradioactive versions of all polypeptides (mock-PET tracers) aretested first to determine if conjugation alters their stability orintegrin binding affinity. This is not expected, since the conjugationchemistry will occur at a site in the polypeptide that is distant fromthe RGD-based integrin binding loop, where prior Cy5.5 conjugation hasshown little effect (FIG. 8).

αvβ3 integrin binding assay: An α_(v)β3 integrin receptor binding assayis performed to determine the relative affinities of the mock-PETtracers compared to their unlabeled polypeptides. Briefly, 2×10⁵ U87MGglioblastoma cells are incubated with 0.06 nM ¹²⁵I-echistatin inintegrin binding buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl₂ 1 mM CaCl₂1 mM MgCl₂, 1 mM MnCl₂, 0.1% BSA), in the presence of increasingconcentrations of mock-PET tracers at room temperature. After incubationfor 3 h, cells are pelleted by centrifugation at 1500 RPM and washedthree times in binding buffer to remove unbound ligands. Theradioactivity remaining in the cell pellet is measured by y counting.IC₅₀ values are determined by plotting the % competition and using afour-point binding equation (Kaleidagraph) to fit the data.

In-vitro serum stability studies: Nonradioactive mock-PET tracers areincubated with mouse and human serum for various time points at 37° C.,and aliquots are acidified and flash frozen. The aliquots are thenthawed and microcentrifuged at high speeds to remove aggregates. Thesoluble fraction is passed through a Sep-Pak C18 cartridge (WatersCorp), and rinsed several times with water containing 0.1% TFA (solventA). The cartridge-bound PET-tracers are eluted with 90% acetonitrilecontaining 0.1% TFA, lyophilized, and resuspended in solvent A. Thesolution is passed through a NANOSEP (Pall Corp) 10 kDa cutoff filterand analyzed by reversed-phase HPLC to determine the amount ofpolypeptide-conjugate remaining.

In-vivo metabolic stability study: The metabolic stability ofradiolabeled PET tracers is evaluated in normal athymic nude mice. Theseanimals are sacrificed and dissected at various time points (30 min, 60min, 120 min) after injection of radiotracer via the tail vein. Blood isimmediately be centrifuged at 15,000 g for 5 min. Liver and kidneys arehomogenized and extracted with phosphate buffered saline (PBS) andcentrifuged at 15,000 g for 5 min. The extracted organ fractions and aurine sample are separately passed through Sep-Pak C18 cartridges(Waters Corp) to collect the radioactive polypeptide tracers. The PETtracers are eluted with 90% acetonitrile containing 0.1% TFA,lyophilized, and resuspended in solvent A. The solution are analyzed byreversed-phase HPLC to determine how much of the tracer is intact postinjection and the clearance half-life from different organs.

MicroPET Imaging in Mouse Tumor Models Using RadiolabeledIntegrin-Binding Polypeptides

To assess the potential of integrin-binding polypeptides as clinicalimaging agents, six polypeptides (c(RGDyK) (SEQ ID NO: 140), FN-RDG,FN-RGD, Miniprotein 1.5B, Miniprotein 2.5D, and Miniprotein 2.5F areconjugated to ¹⁸F or ^(M)Cu. Three polypeptide concentrations aretested, ranging from pmol to nmol. Each imaging study is performed inreplicates with three mice.

U87MG glioblastoma xenoqraft mouse model: All animal procedures areperformed in the Stanford Small Animal Imaging Facility, according toprotocols approved by the Stanford University Administrative Panels onLaboratory Animal Care. The U87MG glioblastoma cell line (ATCC,Manassas, Va.) is maintained at 37° C. in a humidified atmospherecontaining 5% CO₂ in Isocove's modified Dulbecco's medium supplementedwith 5% heat-inactivated fetal bovine serum (Invitrogen Carlsbad Calif.)and penicillin/streptomycin as an antibiotic. Female athymic nude mice(nu/nu) obtained from Charles River Laboratories, Inc (Cambridge, Mass.)4 to 6 weeks of age, are subcutaneously injected on the shoulder with2×10⁷ U87MG glioblastoma cells suspended in 100-uL of phosphate bufferedsaline. Tumors are allowed to grow to approximately 1 cm for themicroPET imaging experiments.

MicroPET imaging: MicroPET imaging of tumor-bearing mice is performed ona microPET R4 rodent model scanner (Concorde Microsystems Inc,Knoxville, Tenn.). U87MG tumor bearing mice are injected with PETimaging agent via the tail vein. At various times post injection, themice are anesthetized with 2% isoflurane, and 10 min static scans areobtained. Images are reconstructed by a two-dimensional orderedexpectation maximum subset algorithm as previously described. Regions ofinterest (ROI) are drawn over the tumor on decay corrected whole bodyimages and ROI derived % injected dose per gram of tissue is determined.Statistical analysis is performed using the student's t-test forunpaired data. A 95% confidence level is used to determine statisticalsignificance.

Female athymic mice bearing U87MG tumors were injected with 80-150 μCiof Cu-DOTA-knottin 2.5D or 7-9 μCi of [¹⁸F]-FB-E[knottin 2.5D]. Staticimages were taken at various timepoints post injection using a microPETR4 rodent model scanner (Concorde Microsystems Inc, Knoxville, Tenn.).Both the ⁶⁴Cu- and ¹⁸F-labeled knottin 2.5D clearly targeted the U87MGtumor, with high contrast relative to the contralateral background.Uptake was also observed in the kidney as both probes cleared throughthe bladder. Probe uptake in the tumor was essentially blocked bypreinjection of the unlabeled peptide demonstrating specific targetingof the tumor.

In vivo biodistribution studies: Mice are sacrificed by exanguinationsat various time points postinjection. Blood, tumor and the major organsand tissues are collected, wet-weighed and measured in a γ-counter. The% injected dose per gram is determined for each sample. For each mouse,radioactivity of the tissue samples is calibrated against a knownaliquot of the injectate. Values are reported as the mean±standarddeviation.

The following Table 10 quantifies the ⁶⁴Cu-DOTA knottin uptake by directtissue sampling of the mice up to 24 hours post injection and isreported in % ID (percent injected dose)/gram. Similar results wereobtained with [¹⁸F]-FB-E[knottin 2.5D].

TABLE 9 Organ Uptake Mean 0.5 h 1 h 2 h 4 h 25 h Tumor 4.94 4.47 2.892.24 1.31 Liver 1.39 1.29 0.98 0.92 0.53 Kidney 7.26 4.06 3.45 3.26 1.75Muscle 0.45 0.28 0.07 0.06 0.03

The above results compare favorably with imaging done with cyclic RGD.

In previous work, glycosylation or polyethylene glycol modification ofthe c(RGDyK) (SEQ ID NO: 140) pentapeptide was shown to enhance itspharmacokinetic profiles compared to the unmodified c(RGDyK) (SEQ ID NO:140) PET tracer. (See, Chen, X., Hou, Y., Tohme, M., Park, R,Khankaldyyan, V., Gonzales-Gomez, I., Bading, J. R, Laug, W. E. & Conti,P. S. (2004). Pegylated Arg-Gly-Asp peptide: ⁶⁴Cu labeling and PETimaging of brain tumor alphavbeta3-integrin expression. J Nucl Med 45,1776-83., and Haubner, R, Wester, H. J., Burkhart, F.,Senekowitsch-Schmidtke, R, Weber, W., Goodman, S. L., Kessler, H. &Schwaiger, M. (2001). Glycosylated RGD-containing peptides: tracer fortumor targeting and angiogenesis imaging with improved biokinetics. JNucl Med 42, 326-36.)

Moreover, [¹⁸F] Galacto-c(RGDfK) has recently been used in humans forPET-based clinical trials, and its uptake was shown to correlate wellwith expression α_(v)β₃ in several different human tumors. (See, Beer, AJ., Haubner, R, Wolf, I., Goebel, M., Luderschmidt, S., Niemeyer, M.,Grosu, A L., Martinez, M. J., Wester, H. J., Weber, W. A & Schwaiger, M.(2006). PET-based human dosimetry of 18F-galacto-RGD, a new radiotracerfor imaging alpha v beta3 expression. J Nucl Med 47,7639., and Haubner,R, Weber, W. A, Beer, A J., Vabuliene, E., Reim, D., Sarbia, M., Becker,K. F., Goebel, M., Hein, R, Wester, H. J., Kessler, H. & Schwaiger, M.(2005). Noninvasive visualization of the activated alpha v beta 3integrin in cancer patients by positron emission tomography and[18F]Galacto-RGD. PLoS Med 2, e70.) Similar polypeptide modificationscan be applied here if PET imaging data indicates poor pharmacokineticsor biodistribution in vivo.

Other Labeling Strategies

Peptides with paramagnetic ions as labels for use in magnetic resonanceimaging have also been described (Lauffer, R. B. Magnetic Resonance inMedicine 1991 22:339-342). The label used will be selected in accordancewith the imaging modality to be used. For example, radioactive labelssuch as Indium-111, Technetium-99m or Iodine-131 can be used for planarscans or single photon emission computed tomography (SPECT). Positronemitting labels such as Fluorine-19 can be used in positron emissiontomography. Paramagnetic ions such as Gadlinium (III) or Manganese (II)can be used in magnetic resonance imaging (MRI). Presence of the label,as compared to imaging of normal tissue, permits determination of thespread of the cancer. The amount of label within an organ or tissue alsoallows determination of the presence or absence of cancer in that organor tissue. ⁹⁰Y and ¹⁷⁷Lu may be used for therapy.

DOTA Chemical Conjugation and ⁶⁴Cu Radiolabeling of Peptides 2.5D and2.5F

Peptides 2.5D and 2.5F, containing the engineered sequences flanking theRGD integrin binding motif, were radiolabeled, as were controlscontaining the native fibronectin RGD sequence and a scrambled RGDsequence (RDG instead of RGD).

The radiolabel was coupled at the amino terminus through1,4,7,10-tetradodecane-N, N′, N″, N′″-tetraacetic acid (DOTA; SigmaAldrich) which was activated with1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC; Pierce) andN-hydroxysulfonosuccinimide (SNHS; Pierce) in water (pH 5.5) for 40 minat room temperature using a 1:1:1 molar ratio of DOTA:EDC:SNHS. Peptideswere dissolved in 300 μL of sodium phosphate buffer (30 mM, pH 8.5), andadded to the above in-situ prepared sulfosuccinimidyl ester of DOTA(DOTA-OSSu). A molar excess of DOTA-OSSu was used to drive theconjugation reaction to completion. The reaction was allowed to proceedat room temperature for 1 h and mixed at 4° C. overnight. The resultingDOTA-peptide conjugates were purified by reversed-phase HPLC and storedas a lyophilized solid. The product masses were verified by ESI-MS andMALDI-TOF-MS and peptide concentrations were determined by amino acidanalysis.

The DOTA-conjugated peptides (25 μg) were radiolabeled with ⁶⁴Cu byincubating with 2-3 mCi ⁶⁴CuCl₂ (University of Wisconsin-Madison,Madison, Wis.) in 0.1 N sodium acetate (pH 6.3) for 1 h at 45° C. Thereaction was terminated with the addition of EDTA. The radiolabeledcomplexes were purified using a PD-10 column (Amersham) or by radio-HPLCusing a gamma detector, dried by rotary evaporation, reconstituted inPBS, and passed through a 0.22 μm filter for animal experiments. Theradiochemical purity, determined as the ratio of the main product peakto other peaks, was determined by HPLC to be >95%. The radiochemicalyield, determined as the ratio of final activity of the product over thestarting activity used for the reaction, was usually over 80%. At least7 radiolabeling reactions were performed for experiments run ondifferent days.

A U87MG glioblastoma xenograft mouse model was used. U87MG cells weremaintained at 37° C. in a humidified atmosphere containing 5% CO₂ inDulbecco's modified eagle medium, 10% heat-inactivated fetal bovineserum, and penicillin-streptomycin (all from Invitrogen). Animalprocedures were carried out according to a protocol by StanfordUniversity Administrative Panels on Laboratory Animal Care. Femaleathymic nude mice (nu/nu), obtained at 4-6 weeks of age (Charles RiverLaboratories, Inc.), were injected subcutaneously in the right or leftshoulder with 2×10⁷ U87MG glioblastoma cells suspended in 100 μL of PBS.Mice were used for in vivo imaging studies when their tumors reachedapproximately 8 to 10 millimeters in diameter.

U87MG tumor-bearing mice (n=3 or more for each probe) were injected with˜100 μCi of Cu-DOTA-conjugated peptides via the tail vein and imagedwith a microPET R4 rodent model scanner (Siemens Medical) using 3 or 5min static scans. For blocking experiments, mice were co-injected with330 μg (˜0.5 μmol) of unlabeled c(RGDyK) (SEQ ID NO: 140). Images werereconstructed by a two dimensional ordered expectation maximum subsetalgorithm and calibrated as previously described (Wu, Y, Zhang, X,Xiong, Z, et al. microPET imaging of glioma integrin {alpha}v{beta}3expression using (64)Cu-labeled tetrameric RGD peptide. J Nucl Med 2005;46:1707-18). ROIs were drawn over the tumor on decay-corrected wholebody images using ASIPro VM software (Siemens Medical). The mean countsper pixel per minute were obtained from the ROI and converted to countsper milliliter per minute with a calibration constant. ROIs wereconverted to counts/g/min, and % ID/g values were determined assuming atissue density of 1 g/mL. No attenuation correction was performed.

Knottin peptides 2.5D and 2.5F were shown to bind to U87MG cells with asignificantly stronger affinity (IC₅₀=19±6 nM and 26±5 nM, respectively)than both the loop-grafted FN-RGD2 (IC₅₀=370±150 nM) and c(RGDyK) (SEQID NO: 140) (IC₅₀=860±400 nM) peptides. FN-RDG2 was not able to competefor ¹²⁵I-echistatin binding to U87MG cells, as expected. Next,DOTA-conjugated peptides were shown to bind to U87MG cells in adose-dependent manner with affinities that were comparable to theunmodified peptides. Since U87MG cells have been shown to expressα_(v)β₅, and α₅β₁ integrins in addition to α_(v)β₃ integrin, we measuredintegrin binding specificity by competition of ¹²⁵I-echistatin todetergent-solubilized α_(v)β₃, α_(v)β₅, α₅β₁, and α_(IIb)β₃ integrinreceptors coated onto microtiter plates. Unlabeled echistatin, ourpositive control, bound strongly to all of the tested integrins, inagreement with previous reports. All RGD-containing peptides bound toα_(v)β₃ and α_(v)β₅ integrins to some degree, with the knottin peptides2.5D, and 2.5F showing the strongest levels of binding compared toFN-RGD2 and c(RGDyK) (SEQ ID NO: 140). DOTA-conjugated FN-RDG2, ournegative control (having the RGD sequence switched to RDG), did not bindto any of the integrins used in this study.

Tumor uptake at 1 h post injection for two high affinity (IC₅₀ ˜20 nM)Cu-DOTA-conjugated knottin peptides was 4.47±1.21 and 4.56±0.64%injected dose/gram (% ID/g), compared to a low affinity knottin peptide(IC₅₀ ˜0.4 μM; 1.48±0.53% ID/g) and c(RGDyK) (SEQ ID NO: 140) (IC₅₀ ˜1μM; 2.32±0.55% ID/g), a low affinity cyclic pentapeptide under clinicaldevelopment. Furthermore, Cu-DOTA-conjugated knottin peptides generatedlower levels of non-specific liver uptake (˜2% ID/g) compared toc(RGDyK) (SEQ ID NO: 140) (˜4% ID/g) 1 h post injection. MicroPETimaging results were confirmed by in vivo biodistribution studies.⁶⁴Cu-DOTA-conjugated knottin peptides were stable in mouse serum, and invivo metabolite analysis showed minimal degradation in the blood ortumor upon injection. Thus, engineered integrin-binding knottin peptidesshow great potential as clinical diagnostics for a variety of cancers.

The above results showed that Cy5.5- (optical label described above) and⁶⁴Cu-DOTA-conjugated FN-RGD2 knottin peptides, which bind to integrinswith affinities in the low micromolar range, generated significantlyweaker imaging signals compared to knottin peptides 2.5D and 2.5F. Theseresults strongly suggest that integrin binding affinity influences tumoruptake of knottin peptides, although other factors such ashydrophobicity can also affect tissue biodistribution. Interestingly, inPET studies knottin peptide 2.5F exhibited slower tumor washout comparedto 2.5D, resulting in much higher tumor/blood ratios 4 h post injection.This could be due to the ability of knottin 2.5F to bind more tightly toα₅β₁ integrins compared to knottin 2.5D or potential differences inpeptide hydrophobicity, charge, or off-rates of integrin receptorbinding. Finally, we demonstrated that knottin peptides were stable invitro upon prolonged serum incubation, and in vivo in the tumor andblood during the timeframe in which imaging experiments were performed.In addition to increased tumor uptake, high affinity ⁶⁴Cu-DOTA-labeledknottin peptides 2.5D and 2.5F demonstrated more favorable tissuedistribution as shown by lower liver uptake compared to⁶⁴Cu-DOTA-c(RGDyK). PEGylated versions of the knottin peptides, as wellas oligomeric knottin proteins that present multiple integrin-bindingRGD motifs may be synthesized according to known methods and used in theimaging application described here. Based on the teachings of thepresent disclosure, it may be expected that these peptides will elicitenhanced tissue distribution and/or tumor uptake compared to unmodifiedknottin peptides, much like that observed with PEGylated and multivalentc(RGDyK) (SEQ ID NO: 140) peptides, respectively.

Imaging with Agouti Peptide 7C Labeled with DOTA-⁶⁴Cu

This example was carried out in similar manner to that above. A ⁶⁴CuLabeled AgRP loop 4 RDG mutant (peptide 7C, described above). It wasshown by HPLC radiochromatogram to be essentially pure. Its bindingactivity was demonstrated with a U87MG cell ¹²⁵I-echistatin competitionbinding assay, as described above. 7C showed better binding than 3F, 6Eor 6F. In vitro cell uptake of the ^(M)Cu labeled 7C on U87-MG cellscould be blocked with cRGDyK (SEQ ID NO: 140). Biodistribution studiesshowed preferential uptake of the labeled 7C by a U87-MG tumor implantedin nude mice. Of non-tumor tissue, the kidneys were shown to have thehighest uptake. These data are shown in FIG. 13.

Peptide Formulations

The present invention also encompasses a pharmaceutical compositionuseful in the treatment of cancer, comprising the administration of atherapeutically effective amount of the compounds of this invention,with or without pharmaceutically acceptable carriers or diluents.Suitable compositions of this invention include aqueous solutionscomprising compounds of this invention and pharmacologically acceptablecarriers, e.g., saline, at a pH level, e.g., 7.4. The solutions may beintroduced into a patient's bloodstream by local bolus injection.

When a compound according to this invention is administered into a humansubject, the daily dosage will normally be determined by the prescribingphysician with the dosage generally varying according to the age,weight, and response of the individual patient, as well as the severityof the patient's symptoms.

In one exemplary application, a suitable amount of compound isadministered to a mammal undergoing treatment for cancer. Administrationoccurs in an amount between about 0.1 mg/kg of body weight to about 60mg/kg of body weight per day, preferably of between 0.5 mg/kg of bodyweight to about 40 mg/kg of body weight per day.

The pharmaceutical composition may be administered parenterally,topically, orally or locally. It is preferably given by parenteral,e.g., subcutaneous, intradermal or intramuscular route, preferably bysubcutaneous or intradermal route, in order to reach proliferating cellsin particular (e.g., potential metastases and tumor cells). Within thescope of tumor therapy the peptide may also be administered directlyinto a tumor.

The composition according to the invention for parenteral administrationis generally in the form of a solution or suspension of the peptide in apharmaceutically acceptable carrier, preferably an aqueous carrier.Examples of aqueous carriers that may be used include water, bufferedwater, saline solution (0.4%), glycine solution (0.3%), hyaluronic acidand similar known carriers. Apart from aqueous carriers it is alsopossible to use solvents such as dimethylsulphoxide, propyleneglycol,dimethylformamide and mixtures thereof. The composition may also containpharmaceutically acceptable excipients such as buffer substances andinorganic salts in order to achieve normal osmotic pressure and/oreffective lyophilization. Examples of such additives are sodium andpotassium salts, e.g., chlorides and phosphates, sucrose, glucose,protein hydrolysates, dextran, polyvinylpyrrolidone or polyethyleneglycol. The compositions may be sterilized by conventional methods,e.g., by sterile filtration. The composition may be decanted directly inthis form or lyophilized and mixed with a sterile solution before use.

In one embodiment, the pharmaceutical composition according to theinvention is in the form of a topical formulation, e.g., for dermal ortransdermal application. The pharmaceutical composition may, forexample, take the form of hydrogel based on polyacrylic acid orpolyacrylamide (such as Dolobene®, Merckle), as an ointment, e.g., withpolyethyleneglycol (PEG) as the carrier, like the standard ointment DAB8 (50% PEG 300, 50% PEG 1500), or as an emulsion, especially amicroemulsion based on water-in-oil or oil-in-water, optionally withadded liposomes. Suitable permeation accelerators (entraining agents)include sulphoxide derivatives such as dimethylsulphoxide (DMSO) ordecylmethylsulphoxide (decyl-MSO) and transcutol(diethyleneglycolmonoethylether) or cyclodextrin, as well aspyrrolidones, e.g., 2-pyrrolidone, N-methyl-2-pyrrolidone,2-pyrrolidone-5-carboxylic acid or the biodegradableN-(2-hydroxyethyl)-2-pyrrolidone and the fatty acid esters thereof, ureaderivatives such as dodecylurea, 1,3-didodecylurea and 1,3-diphenylurea,terpenes, e.g., D-limonene, menthone, a-terpinol, carvol, limonene oxideor 1,8-cineol.

Other formulations are aerosols, e.g., for administering as a nasalspray or for inhalation.

The composition according to the invention may also be administered bymeans of liposomes which may take the form of emulsions, foams,micelles, insoluble monolayers, phospholipid dispersions, lamella layersand the like. These act as carriers for conveying the peptides to theirtarget of a certain tissue, e.g., lymphoid tissue or tumor tissue or toincrease the half-life of the peptides. The present peptides may also beformulated for oral peptide delivery, e.g., with organic acids toinactivate digestive enzymes and a detergent, or bile acid fortemporarily opening up the tight junctions within the intestine tofacilitate transport into the bloodstream. The present peptides may alsobe conjugated to carriers such as polyethylene glycol, or modified byglycosylation, or acylation for improvement of circulatory half-life.

If the composition according to the invention is in the form of atopical formulation it may also contain UV-absorbers in order to act,for example, as a sun protection cream, for example, when theformulation is used prophylactically against melanoma.

The person skilled in the art will find suitable formulations andadjuvants in standard works such as “Remington's PharmaceuticalSciences,” 1990.

CONCLUSION

The above specific description is meant to exemplify and illustrate theinvention and should not be seen as limiting the scope of the invention,which is defined by the literal and equivalent scope of the appendedclaims. Any patents or publications mentioned in this specification,including the below cited references are indicative of levels of thoseskilled in the art to which the patent pertains and are intended toconvey details of the invention which may not be explicitly set out butwhich would be understood by workers in the field Such patents orpublications are hereby incorporated by reference to the same extent asif each was specifically and individually incorporated by reference, asneeded for the purpose of describing and enabling the methods andmaterials.

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What is claimed is:
 1. A pharmaceutical composition, comprising: anintegrin binding peptide comprising a knottin protein scaffoldcomprising an engineered integrin binding loop that binds to at leastone of αvβ5 integrin, αvβ3 integrin and α5β1 integrin, wherein theintegrin binding peptide comprises an amino acid sequence at least 90%identical to the amino acid sequence of a peptide of any one of SEQ IDNO:23 through SEQ ID NO:52; and a pharmaceutically acceptable carrier.2. The pharmaceutical composition of claim 1, wherein the integrinbinding peptide comprises an amino acid sequence at least 95% identicalto the amino acid sequence of a peptide of any one of SEQ ID NO:23through SEQ ID NO:52.
 3. The pharmaceutical composition of claim 1,wherein the integrin binding peptide comprises the amino acid sequenceof a peptide of any one of SEQ ID NO:23 through SEQ ID NO:52.
 4. Thepharmaceutical composition of claim 1, wherein the integrin bindingpeptide comprises the amino acid sequence of the peptide of SEQ IDNO:49.
 5. The pharmaceutical composition of claim 1, wherein theintegrin binding peptide comprises the amino acid sequence of thepeptide of SEQ ID NO:50.
 6. The pharmaceutical composition of claim 1,wherein the integrin binding peptide has a Kd of not more than 100 nM.7. The pharmaceutical composition of claim 1, wherein the integrinbinding peptide binds to two or more of αvβ5 integrin, αvβ3 integrin andα5β1 integrin.
 8. The pharmaceutical composition of claim 1, wherein theintegrin binding peptide is conjugated to a chemotherapeutic agent. 9.The pharmaceutical composition of claim 1, wherein the integrin bindingpeptide is conjugated to a half-life extending moiety.
 10. Thepharmaceutical composition of claim 1, wherein the composition issuitable for parenteral, oral, topical, or local administration to asubject.
 11. A pharmaceutical composition, comprising: a peptide thatbinds to at least one of αvβ5 integrin, αvβ3 integrin and α5β1 integrin,the peptide comprising a scaffold portion as set forth in SEQ ID NO: 19and a binding loop portion, wherein, in the binding loop portion: X1 isselected from the group consisting of A, V, L, P, F, Y, S, H, D, and N;X2 is selected from the group consisting of G, V, L, P, R, E, and Q; X3is selected from the group consisting of G, A, and P; X7 is selectedfrom the group consisting of W and N; X8 is selected from the groupconsisting of A, P, and S; X9 is selected from the group consisting of Pand R; X10 is selected from the group consisting of A, V, L, P, S, T,and E; and X11 is selected from the group consisting of G, A, W, S, T,K, and E, and a pharmaceutically acceptable carrier.
 12. Thepharmaceutical composition of claim 11, wherein, in the binding loopportion: X1 is P; X2 is Q or R; X3 is G or P; X7 is W or N; X8 is A orP; X9 is P; X10 is T or L; and X11 is S or T.
 13. The pharmaceuticalcomposition of claim 11, wherein, in the binding loop portion: X1 is P;X2 is Q; X3 is G; X7 is W; X8 is A; X9 is P; X10 is T; and X11 is S. 14.The pharmaceutical composition of claim 11, wherein, in the binding loopportion: X1 is P; X2 is R; X3 is P; X7 is N; X8 is P; X9 is P; X10 is L;and X11 is T.
 15. The pharmaceutical composition of claim 11, whereinthe integrin binding peptide has a Kd of not more than 100 nM.
 16. Thepharmaceutical composition of claim 11, wherein the integrin bindingpeptide binds to two or more of αvβ5 integrin, αvβ3 integrin and α5β1integrin.
 17. The pharmaceutical composition of claim 11, wherein theintegrin binding peptide is conjugated to a chemotherapeutic agent or ahalf-life extending moiety.
 18. The pharmaceutical composition of claim11, wherein the composition is suitable for parenteral, oral, topical,or local administration to a subject.
 19. A method, comprising:administering to a subject the pharmaceutical composition of claim 1.20. A method, comprising: administering to a subject the pharmaceuticalcomposition of claim 11.